Methods for identifying factors for differentiating definitive endoderm

ABSTRACT

Disclosed herein are methods of identifying one or more differentiation factors that are useful for differentiating cells in a cell population comprising definitive endoderm cells into cells which are capable of forming tissues and/or organs that are derived from the gut tube.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/115,868, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 26,2005, now abandoned which claims priority under 35 U.S.C. § 119(e) as anonprovisional application of U.S. Provisional Patent Application No.60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S.Provisional Patent Application No. 60/587,942, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul.14, 2004; and U.S. Provisional Patent Application No. 60/586,566,entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM, filed Jul. 9, 2004; this application is also acontinuation-in-part of U.S. patent application Ser. No. 11/021,618,entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, now U.S. Pat. No.7,510,876 which claims priority under 35 U.S.C. § 119(e) as anonprovisional application to U.S. Provisional Patent Application No.60/587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATIONOF DEFINITIVE ENDODERM, filed Jul. 14, 2004; and U.S. Provisional PatentApplication No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FORTHE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004. and U.S.Provisional Patent Application No. 60/532,004, entitled DEFINITIVEENDODERM, filed Dec. 23, 2003. The disclosure of each of the foregoingpriority applications is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cellbiology. In particular, the present invention relates the identificationof factors that are useful for differentiating definitive endoderm cellsinto other cell types.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells andembryonic germ (EG) cells, were first isolated in culture withoutfibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblastfeeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblottestablished continuous cultures of human ES and EG cells usingmitotically inactivated mouse feeder layers (Reubinoff et al., 2000;Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities forinvestigating early stages of human development as well as fortherapeutic intervention in several disease states, such as diabetesmellitus and Parkinson's disease. For example, the use ofinsulin-producing β-cells derived from hESCs would offer a vastimprovement over current cell therapy procedures that utilize cells fromdonor pancreases for the treatment of diabetes. However, presently it isnot known how to generate an insulin-producing β-cell from hESCs. Assuch, current cell therapy treatments for diabetes mellitus, whichutilize islet cells from donor pancreases, are limited by the scarcityof high quality islet cells needed for transplant. Cell therapy for asingle Type I diabetic patient requires a transplant of approximately8×10⁸ pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al.,2001a; Shapiro et al., 2001b). As such, at least two healthy donororgans are required to obtain sufficient islet cells for a successfultransplant. Human embryonic stem cells offer a source of startingmaterial from which to develop substantial quantities of high qualitydifferentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapyapplications are pluripotence and the ability to maintain these cells inculture for prolonged periods. Pluripotency is defined by the ability ofhESCs to differentiate to derivatives of all 3 primary germ layers(endoderm, mesoderm, ectoderm) which, in turn, form all somatic celltypes of the mature organism in addition to extraembryonic tissues (e.g.placenta) and germ cells. Although pluripotency imparts extraordinaryutility upon hESCs, this property also poses unique challenges for thestudy and manipulation of these cells and their derivatives. Owing tothe large variety of cell types that may arise in differentiating hESCcultures, the vast majority of cell types are produced at very lowefficiencies. Additionally, success in evaluating production of anygiven cell type depends critically on defining appropriate markers.Achieving efficient, directed differentiation is of great importance fortherapeutic application of hESCs.

In order to use hESCs as a starting material to generate cells that areuseful in cell therapy applications, it would be advantageous toovercome the foregoing problems. Additionally, it would be beneficial toidentify factors which promote the differentiation of precursor cellsderived from hESCs to cell types useful for cell therapies.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to methods of identifyingone or more differentiation factors that are useful for differentiatingcells in a cell population comprising PDX1-positive (PDX1-expressing)endoderm cells and/or PDX1-negative endoderm cells (endoderm cells whichdo not significantly express PDX1), such as definitive endoderm cells,into cells that are useful for cell therapy. For example, someembodiments of the methods described herein relate to methods ofidentifying factors capable of promoting the differentiation ofdefinitive endoderm cells into cells which are precursors for tissuesand/or organs which include, but are not limited to, pancreas, liver,lungs, stomach, intestine, thyroid, thymus, pharynx, gallbladder andurinary bladder. In some embodiments, such precursor cells arePDX1-positive endoderm cells. In other embodiments, such precursor cellsare endoderm cells that do not significantly express PDX1.

In some embodiments of the methods described herein, cell cultures orcell populations of definitive endoderm cells are contacted or otherwiseprovided with a candidate (test) differentiation factor. In preferredembodiments, the definitive endoderm cells are human definitive endodermcells. In more preferred embodiments, the human definitive endodermcells are multipotent cells that can differentiate into cells of the guttube or organs derived therefrom.

In other embodiments of the methods described herein, cell cultures orcell populations of PDX1-positive endoderm cells are contacted orotherwise provided with a candidate differentiation factor. In preferredembodiments, the PDX1-positive endoderm cells are human PDX1-positiveendoderm cells. In certain embodiments, the human PDX1-positive endodermcells are PDX1-positive foregut/midgut endoderm cells. In more preferredembodiments, the human PDX1-positive endoderm cells are PDX1-positiveforegut endoderm cells. In other preferred embodiments, the humanPDX1-positive endoderm cells are PDX1-positive endoderm cells of theposterior portion of the foregut. In especially preferred embodiments,the human PDX1-positive foregut endoderm cells are multipotent cellsthat can differentiate into cells, tissues or organs derived from theanterior portion of the gut tube.

As related to the methods described herein, the candidatedifferentiation factor may be one that is known to cause celldifferentiation or one that is not known to cause cell differentiation.In certain embodiments, the candidate differentiation factor can be apolypeptide, such as a growth factor. In some embodiments, the growthfactor includes, but is not limited to, FGF10, FGF4, FGF2, Wnt3A orWnt3B. In other embodiments, the candidate differentiation factor can bea small molecule. In particular embodiments, the small molecule is aretinoid compound, such as retinoic acid. Alternatively, in someembodiments, the candidate differentiation factor is not a retinoid, isnot a foregut differentiation factor or is not a member of the TGFβsuperfamily. In other embodiments, the candidate differentiation factoris any molecule other than a retinoid compound, a foregutdifferentiation factor, or a member of the TGFβ superfamily of growthfactors, such as activins A and B. In still other embodiments, thecandidate differentiation factor is a factor that was not previouslyknown to cause the differentiation of definitive endoderm cells.

Additional embodiments of the methods described herein relate to testingcandidate differentiation factors at a plurality of concentrations. Forexample, a candidate differentiation factor may cause thedifferentiation of definitive endoderm cells and/or PDX1-positiveendoderm cells only at concentrations above a certain threshold.Additionally, a candidate differentiation factor can cause the same cellto differentiate into a first cell type when provided at a lowconcentration and a second cell type when provided at a higherconcentration. In some embodiments, the candidate differentiation factoris provided at one or more concentrations ranging from about 0.1 ng/mlto about 10 mg/ml.

Prior to or at approximately the same time as contacting or otherwiseproviding the cell culture or cell population comprising definitiveendoderm cells and/or PDX1-positive endoderm cells with the candidatedifferentiation factor, at least one marker is selected and evaluated soas to determine its expression. This step may be referred to as thefirst marker evaluation step. Alternatively, this step may be referredto as determining expression of a marker at a first time point. Themarker can be any marker that is useful for monitoring celldifferentiation, however, preferred markers include, but are not limitedto, sex determining region Y-box 17 (SOX17), pancreatic-duodenalhomeobox factor-1 (PDX1), albumin, hepatocyte specific antigen (HAS),prospero-related homeobox 1 (PROX1), thyroid transcription factor 1(TITF1), villin, alpha fetoprotein (AFP), cytochrome P450 7A (CYP7A),tyrosine aminotransferase (TAT), hepatocyte nuclear factor 4a (HNF4a),CXC-type chemokine receptor 4 (CXCR4), von Willebrand factor (VWF),vascular cell adhesion molecule-1 (VACM1), apolipoprotein A1 (APOA1),glucose transporter-2 (GLUT2), alpha-1-antitrypsin (AAT), glukokinase(GLUKO), and human hematopoietically expressed homeobox (hHEX) and CDX2.

After sufficient time has passed since contacting or otherwise providingcell culture or cell population comprising definitive endoderm cellsand/or PDX1-positive endoderm cells with the candidate differentiationfactor, the expression of the at least one marker in the cell culture orcell population is again evaluated. This step may be referred to as thesecond marker evaluation step. Alternatively, this step may be referredto as determining expression of a marker at a second time point. Inpreferred embodiments, the marker evaluated at the first and second timepoints is the same marker.

In some embodiments of the methods described herein, it is furtherdetermined whether the expression of the at least one marker at thesecond time point has increased or decreased as compared to theexpression of this marker at the first time point. An increase ordecrease in the expression of the at least one marker indicates that thecandidate differentiation factor is capable of promoting thedifferentiation of the definitive endoderm cells and/or thePDX1-positive endoderm cells. Sufficient time between contacting orotherwise providing a cell culture or cell population comprisingdefinitive endoderm cells and/or PDX1-positive endoderm cells with thecandidate differentiation factor and determining expression of the atleast one marker at the second time point can be as little as from about1 hour to as much as about 10 days. In some embodiments, the expressionof the at least one marker is evaluated multiple times subsequent tocontacting or otherwise providing the cell culture or cell populationcomprising definitive endoderm cells and/or PDX1-positive endoderm cellswith the candidate differentiation factor. In certain embodiments,marker expression is evaluated by Q-PCR. In other embodiments, markerexpression is evaluated by immunocytochemistry.

Additional embodiments of the present invention relate to a method ofidentifying a differentiation factor capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In such methods, PDX1-negativedefinitive endoderm cells are contacted with a candidate differentiationfactor and it is determined whether PDX1 expression in the cellpopulation after contact with the candidate differentiation factor hasincreased as compared to PDX1 expression in the cell population beforecontact with the candidate differentiation factor. An increase in thePDX1 expression in the cell population indicates that the candidatedifferentiation factor is capable of promoting the differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells. In some embodiments, PDX1 expression is determined byquantitative polymerase chain reaction (Q-PCR). Some embodiments of theforegoing method further comprise the step of determining expression ofthe HOXA13 and/or the HOXC6 gene in the cell population before and aftercontact with the candidate differentiation factor. In some embodiments,the candidate differentiation factor is a small molecule, for example, aretinoid, such as RA. In others, the candidate differentiation factor isa polypeptide, for example, a growth factor, such as FGF-10.

Still other embodiments of the present invention relate to a method ofidentifying a differentiation factor capable of promoting thedifferentiation of PDX1-positive foregut endoderm cells. In suchmethods, PDX1-positive foregut endoderm cells are contacted with acandidate differentiation factor and it is determined whether expressionof a marker in the population is increased or decreased after contactwith the candidate differentiation factor as compared to the expressionof the same marker in the population before contact with the candidatedifferentiation factor. An increase or decrease in the expression of themarker indicates that the candidate differentiation factor is capable ofpromoting the differentiation of PDX1-positive foregut endoderm cells.In some embodiments, marker expression is determined by Q-PCR. In someembodiments, the candidate differentiation factor is a small molecule,for example, a retinoid, such as RA. In others, the candidatedifferentiation factor is a polypeptide, for example, a growth factor,such as FGF-10.

Yet other embodiments of the present invention relate to cellsdifferentiated by the methods described herein. Such cells include butare not limited to precursors of the pancreas, liver, lungs, stomach,intestine, thyroid, thymus, pharynx, gallbladder and urinary bladder. Insome embodiments, the cells may be terminally differentiated. Otherembodiments described herein relate to cell cultures and/or cellpopulations comprising the above-described cells.

In certain jurisdictions, there may not be any generally accepteddefinition of the term “comprising.” As used herein, the term“comprising” is intended to represent “open” language which permits theinclusion of any additional elements. With this in mind, additionalembodiments of the present inventions are described with reference tothe numbered paragraphs below:

-   -   1. A method of identifying a differentiation factor capable of        promoting the differentiation of human definitive endoderm cells        in a cell population comprising human cells, said method        comprising the steps of: (a) obtaining a cell population        comprising human definitive endoderm cells; (b) providing a        candidate differentiation factor to said cell population; (c)        determining expression of a marker in said cell population at a        first time point; (d) determining expression of the same marker        in said cell population at a second time point, wherein said        second time point is subsequent to said first time point and        wherein said second time point is subsequent to providing said        cell population with said candidate differentiation factor;        and (e) determining if expression of the marker in said cell        population at said second time point is increased or decreased        as compared to the expression of the marker in said cell        population at said first time point, wherein an increase or        decrease in expression of said marker in said cell population        indicates that said candidate differentiation factor is capable        of promoting the differentiation of said human definitive        endoderm cells.    -   2. The method of paragraph 1, wherein said human definitive        endoderm cells comprise at least about 10% of the human cells in        said cell population.    -   3. The method of paragraph 1, wherein human feeder cells are        present in said cell population and wherein at least about 10%        of the human cells other than said feeder cells are definitive        endoderm cells.    -   4. The method of paragraph 1, wherein said human definitive        endoderm cells comprise at least about 90% of the human cells in        said cell population.    -   5. The method of paragraph 1, wherein said human feeder cells        are present in said cell population and wherein at least about        90% of the human cells other than said feeder cells are        definitive endoderm cells.    -   6. The method of paragraph 1, wherein said human definitive        endoderm cells differentiate into cells, tissues or organs        derived from the gut tube in response to said candidate        differentiation factor.    -   7. The method of paragraph 1, wherein said human definitive        endoderm cells differentiate into pancreatic precursor cells in        response to said candidate differentiation factor.    -   8. The method of paragraph 7, wherein said marker is selected        from the group consisting of pancreatic-duodenal homeobox        factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).    -   9. The method of paragraph 1, wherein said human definitive        endoderm cells differentiate into liver precursor cells in        response to said candidate differentiation factor.    -   10. The method of paragraph 9, wherein said marker is selected        from the group consisting of albumin, prospero-related homeobox        1 (PROX1) and hepatocyte specific antigen (HSA).    -   11. The method of paragraph 1, wherein said human definitive        endoderm cells differentiate into lung precursor cells in        response to said candidate differentiation factor.    -   12. The method of paragraph 11, wherein said marker is thyroid        transcription factor 1 (TITF1).    -   13. The method of paragraph 1, wherein said human definitive        endoderm cells differentiate into intestinal precursor cells in        response to said candidate differentiation factor.    -   14. The method of paragraph 13, wherein said marker is selected        from the group consisting of villin and caudal type homeobox        transcription factor 2 (CDX2).    -   15. The method of paragraph 1, wherein said first time point is        prior to providing said candidate differentiation factor to said        cell population.    -   16. The method of paragraph 1, wherein said first time point is        at approximately the same time as providing said candidate        differentiation factor to said cell population.    -   17. The method of paragraph 1, wherein said first time point is        subsequent to providing said candidate differentiation factor to        said cell population.    -   18. The method of paragraph 1, wherein expression of said marker        is increased.    -   19. The method of paragraph 1, wherein expression of said marker        is decreased.    -   20. The method of paragraph 1, wherein expression of said marker        is determined by quantitative polymerase chain reaction (Q-PCR).    -   21. The method of paragraph 1, wherein expression of said marker        is determined by immunocytochemistry.    -   22. The method of paragraph 1, wherein said marker is selected        from the group consisting of pancreatic-duodenal homeobox        factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).    -   23. The method of paragraph 1, wherein said marker is selected        from the group consisting of albumin, prospero-related homeobox        1 (PROX1) and hepatocyte specific antigen (HSA).    -   24. The method of paragraph 1, wherein said marker is selected        from the group consisting of villin and caudal type homeobox        transcription factor 2 (CDX2).    -   25. The method of paragraph 1, wherein said marker is thyroid        transcription factor 1 TITF1).    -   26. The method of paragraph 1, wherein said differentiation        factor comprises a foregut differentiation factor.    -   27. The method of paragraph 1, wherein said differentiation        factor comprises a small molecule.    -   28. The method of paragraph 1, wherein said differentiation        factor comprises a retinoid.    -   29. The method of paragraph 1, wherein said differentiation        factor comprises retinoic acid.    -   30. The method of paragraph 1, wherein said differentiation        factor comprises a polypeptide.    -   31. The method of paragraph 1, wherein said differentiation        factor comprises a growth factor.    -   32. The method of paragraph 1, wherein said differentiation        factor comprises FGF-10.    -   33. The method of paragraph 1, wherein said differentiation        factor comprises FGF-2.    -   34. The method of paragraph 1, wherein said differentiation        factor comprises Wnt3B.    -   35. The method of paragraph 1, wherein said differentiation        factor is not a foregut differentiation factor.    -   36. The method of paragraph 1, wherein said differentiation        factor is not a retinoid.    -   37. The method of paragraph 1, wherein said differentiation        factor is not retinoic acid.    -   38. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        between about 0.1 ng/ml to about 10 mg/ml.    -   39. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        between about 1 ng/ml to about 1 mg/ml    -   40. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        between about 10 ng/ml to about 100 μg/ml.    -   41. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        between about 100 ng/ml to about 10 μg/ml.    -   42. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        about 1 μg/ml.    -   43. The method of paragraph 1, wherein said differentiation        factor is provided to said cell population at a concentration of        about 100 ng/ml.

It will be appreciated that the methods and compositions described aboverelate to cells cultured in vitro. However, the above-described in vitrodifferentiated cell compositions may be used for in vivo applications.

Additional embodiments of the present invention may also be found inU.S. Provisional Patent Application No. 60/532,004, entitled DEFINITIVEENDODERM, filed Dec. 23, 2003; U.S. Provisional Patent Application No.60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S.Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELLSURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9,2004; U.S. Provisional Patent Application No. 60/587,942, entitledCHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVEENDODERM, filed Jul. 14, 2004; U.S. patent application Ser. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, and U.S.patent application Ser. No. 11/115,868, entitled PDX1 EXPRESSINGENDODERM, filed Apr. 26, 2005 the disclosures of which are incorporatedherein by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a proposed differentiation pathway for theproduction of beta-cells from hESCs. The first step in the pathwaycommits the ES cell to the definitive endoderm lineage and alsorepresents the first step prior to further differentiation events topancreatic endoderm, endocrine endoderm, or islet/beta-cells. The secondstep in the pathway shows the conversion of SOX17-positive/PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm. Some factorsuseful for mediating these transitions are italicized. Relevant markersfor defining the target cells are underlined.

FIG. 2 is a diagram of the human SOX17 cDNA which displays the positionsof conserved motifs and highlights the region used for the immunizationprocedure by GENOVAC.

FIG. 3 is a relational dendrogram illustrating that SOX17 is mostclosely related to SOX7 and somewhat less to SOX18. The SOX17 proteinsare more closely related among species homologs than to other members ofthe SOX group F subfamily within the same species.

FIG. 4 is a Western blot probed with the rat anti-SOX17 antibody. Thisblot demonstrates the specificity of this antibody for human SOX17protein over-expressed in fibroblasts (lane 1) and a lack ofimmunoreactivity with EGFP (lane 2) or the most closely related SOXfamily member, SOX7 (lane 3).

FIGS. 5A-B are micrographs showing a cluster of SOX17⁺ cells thatdisplay a significant number of AFP⁺ co-labeled cells (A). This is instriking contrast to other SOX17⁺ clusters (B) where little or no AFP⁺cells are observed.

FIGS. 6A-C are micrographs showing parietal endoderm and SOX17. Panel Ashows immunocytochemistry for human Thrombomodulin (TM) protein locatedon the cell surface of parietal endoderm cells in randomlydifferentiated cultures of hES cells. Panel B is the identical fieldshown in A double-labeled for TM and SOX17. Panel C is the phasecontrast image of the same field with DAPI labeled nuclei. Note thecomplete correlation of DAPI labeled nuclei and SOX17 labeling.

FIGS. 7A-B are bar charts showing SOX17 gene expression by quantitativePCR (Q-PCR) and anti-SOX17 positive cells by SOX17-specific antibody.Panel A shows that activin A increases SOX17 gene expression whileretinoic acid (RA) strongly suppresses SOX17 expression relative to theundifferentiated control media (SR20). Panel B shows the identicalpattern as well as a similar magnitude of these changes is reflected inSOX17⁺ cell number, indicating that Q-PCR measurement of SOX17 geneexpression is very reflective of changes at the single cell level.

FIG. 8A is a bar chart which shows that a culture of differentiatinghESCs in the presence of activin A maintains a low level of AFP geneexpression while cells allowed to randomly differentiate in 10% fetalbovine serum (FBS) exhibit a strong upregulation of AFP. The differencein expression levels is approximately 7-fold.

FIGS. 8B-C are images of two micrographs showing that the suppression ofAFP expression by activin A is also evident at the single cell level asindicated by the very rare and small clusters of AFP⁺ cells observed inactivin A treatment conditions (bottom) relative to 10% FBS alone (top).

FIGS. 9A-B are comparative images showing the quantitation of the AFP⁺cell number using flow cytometry. This figure demonstrates that themagnitude of change in AFP gene expression (FIG. 8A) in the presence(right panel) and absence (left panel) of activin A exactly correspondsto the number of AFP⁺ cells, further supporting the utility of Q-PCRanalyses to indicate changes occurring at the individual cell level.

FIGS. 10A-F are micrographs which show that exposure of hESCs to nodal,activin A and activin B (NAA) yields a striking increase in the numberof SOX17⁺ cells over the period of 5 days (A-C). By comparing to therelative abundance of SOX17⁺ cells to the total number of cells presentin each field, as indicated by DAPI stained nuclei (D-F), it can be seenthat approximately 30-50% of all cells are immunoreactive for SOX17after five days treatment with NAA.

FIG. 11 is a bar chart which demonstrates that activin A (0, 10, 30 or100 ng/ml) dose-dependently increases SOX17 gene expression indifferentiating hESCs. Increased expression is already robust after 3days of treatment on adherent cultures and continues through subsequent1, 3 and 5 days of suspension culture as well.

FIGS. 12A-C are bar charts which demonstrate the effect of activin A onthe expression of MIXL1 (panel A), GATA4 (panel B) and HNF3b (panel C).Activin A dose-dependent increases are also observed for three othermarkers of definitive endoderm; MIXL1, GATA4 and HNF3b. The magnitudesof increased expression in response to activin dose are strikinglysimilar to those observed for SOX17, strongly indicating that activin Ais specifying a population of cells that co-express all four genes(SOX17⁺, MIXL1⁺, GATA4⁺ and HNF3b⁺).

FIGS. 13A-C are bar charts which demonstrate the effect of activin A onthe expression of AFP (panel A), SOX7 (panel B) and SPARC (panel C).There is an activin A dose-dependent decrease in expression of thevisceral endoderm marker AFP. Markers of primitive endoderm (SOX7) andparietal endoderm (SPARC) remain either unchanged or exhibit suppressionat some time points indicating that activin A does not act to specifythese extra-embryonic endoderm cell types. This further supports thefact that the increased expression of SOX17, MIXL1, GATA4, and HNF3b aredue to an increase in the number of definitive endoderm cells inresponse to activin A.

FIGS. 14A-B are bar charts showing the effect of activin A on ZIC1(panel A) and Brachyury expression (panel B) Consistent expression ofthe neural marker ZIC1 demonstrates that there is not a dose-dependenteffect of activin A on neural differentiation. There is a notablesuppression of mesoderm differentiation mediated by 100 ng/ml of activinA treatment as indicated by the decreased expression of brachyury. Thisis likely the result of the increased specification of definitiveendoderm from the mesendoderm precursors. Lower levels of activin Atreatment (10 and 30 ng/ml) maintain the expression of brachyury atlater time points of differentiation relative to untreated controlcultures.

FIGS. 15A-B are micrographs showing decreased parietal endodermdifferentiation in response to treatment with activins. Regions ofTM^(hi) parietal endoderm are found through the culture (A) whendifferentiated in serum alone, while differentiation to TM⁺cells isscarce when activins are included (B) and overall intensity of TMimmunoreactivity is lower.

FIGS. 16A-D are micrographs which show marker expression in response totreatment with activin A and activin B. hESCs were treated for fourconsecutive days with activin A and activin B and triple labeled withSOX17, AFP and TM antibodies. Panel A—SOX17; Panel B—AFP; Panel C—TM;and Panel D—Phase/DAPI. Notice the numerous SOX17 positive cells (A)associated with the complete absence of AFP (B) and TM (C)immunoreactivity.

FIG. 17 is a micrograph showing the appearance of definitive endodermand visceral endoderm in vitro from hESCs. The regions of visceralendoderm are identified by AFP^(hi)/SOX17^(lo/−) while definitiveendoderm displays the complete opposite profile, SOX17^(hi)/AFP^(lo/−).This field was selectively chosen due to the proximity of these tworegions to each other. However, there are numerous times whenSOX17^(hi)/AFP^(lo/−) regions are observed in absolute isolation fromany regions of AFP^(hi) cells, suggesting the separate origination ofthe definitive endoderm cells from visceral endoderm cells.

FIG. 18 is a diagram depicting the TGFβ family of ligands and receptors.Factors activating AR Smads and BR Smads are useful in the production ofdefinitive endoderm from human embryonic stem cells (see, J CellPhysiol. 187:265-76).

FIG. 19 is a bar chart showing the induction of SOX17 expression overtime as a result of treatment with individual and combinations of TGFβfactors.

FIG. 20 is a bar chart showing the increase in SOX17⁺ cell number withtime as a result of treatment with combinations of TGFβ factors.

FIG. 21 is a bar chart showing induction of SOX17 expression over timeas a result of treatment with combinations of TGFβ factors.

FIG. 22 is a bar chart showing that activin A induces a dose-dependentincrease in SOX17⁺ cell number.

FIG. 23 is a bar chart showing that addition of Wnt3a to activin A andactivin B treated cultures increases SOX17 expression above the levelsinduced by activin A and activin B alone.

FIGS. 24A-C are bar charts showing differentiation to definitiveendoderm is enhanced in low FBS conditions. Treatment of hESCs withactivins A and B in media containing 2% FBS (2AA) yields a 2-3 timesgreater level of SOX17 expression as compared to the same treatment in10% FBS media (10 AA) (panel A). Induction of the definitive endodermmarker MIXL1 (panel B) is also affected in the same way and thesuppression of AFP (visceral endoderm) (panel C) is greater in 2% FBSthan in 10% FBS conditions.

FIGS. 25A-D are micrographs which show SOX17⁺ cells are dividing inculture. SOX17 immunoreactive cells are present at the differentiatingedge of an hESC colony (C, D) and are labeled with proliferating cellnuclear antigen (PCNA) (panel B) yet are not co-labeled with OCT4 (panelC). In addition, clear mitotic figures can be seen by DAPI labeling ofnuclei in both SOX17⁺ cells (arrows) as well as OCT4⁺, undifferentiatedhESCs (arrowheads) (D).

FIG. 26 is a bar chart showing the relative expression level of CXCR4 indifferentiating hESCs under various media conditions.

FIGS. 27A-D are bar charts that show how a panel of definitive endodermmarkers share a very similar pattern of expression to CXCR4 across thesame differentiation treatments displayed in FIG. 26.

FIGS. 28A-E are bar charts showing how markers for mesoderm (BRACHYURY,MOX1), ectoderm (SOX1, ZIC1) and visceral endoderm (SOX7) exhibit aninverse relationship to CXCR4 expression across the same treatmentsdisplayed in FIG. 26.

FIGS. 29A-F are micrographs that show the relative difference in SOX17immunoreactive cells across three of the media conditions displayed inFIGS. 26-28.

FIGS. 30A-C are flow cytometry dot plots that demonstrate the increasein CXCR4⁺ cell number with increasing concentration of activin A addedto the differentiation media.

FIGS. 31A-D are bar charts that show the CXCR4⁺ cells isolated from thehigh dose activin A treatment (A100-CX+) are even further enriched fordefinitive endoderm markers than the parent population (A100).

FIG. 32 is a bar chart showing gene expression from CXCR4⁺and CXCR4⁻cells isolated using fluorescence-activated cell sorting (FACS) as wellas gene expression in the parent populations. This demonstrates that theCXCR4⁺ cells contain essentially all the CXCR4 gene expression presentin each parent population and the CXCR4⁻ populations contain very littleor no CXCR4 gene expression.

FIGS. 33A-D are bar charts that demonstrate the depletion of mesoderm(BRACHYURY, MOX1), ectoderm (ZIC1) and visceral endoderm (SOX7) geneexpression in the CXCR4+ cells isolated from the high dose activin Atreatment which is already suppressed in expression of thesenon-definitive endoderm markers.

FIGS. 34A-M are bar charts showing the expression patterns of markergenes that can be used to identify definitive endoderm cells. Theexpression analysis of definitive endoderm markers, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1 is shown in panels G-L, respectively. Theexpression analysis of previously described lineage marking genes,SOX17, SOX7, SOX17/SOX7, TM, ZIC1, and MOX1 is shown in panels A-F,respectively. Panel M shows the expression analysis of CXCR4. Withrespect to each of panels A-M, the column labeled hESC indicates geneexpression from purified human embryonic stem cells; 2NF indicates cellstreated with 2% FBS, no activin addition; 0.1A100 indicates cellstreated with 0.1% FBS, 100 ng/ml activin A; 1A100 indicates cellstreated with 1% FBS, 100 ng/ml activin A; and 2A100 indicates cellstreated with 2% FBS, 100 ng/ml activin A.

FIG. 35 is a chart which shows the relative expression of the PDX1 genein a culture of hESCs after 4 days and 6 days with and without activinin the presence of retinoic acid (RA) and fibroblast growth factor-10(FGF-10) added on day 4.

FIGS. 36A-F are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 6 days with and withoutactivin in the presence of retinoic acid (RA) and fibroblast growthfactor-10 (FGF-10) added on day 4. The panels show the relative levelsof expression of the following marker genes: (A) SOX17; (B) SOX7; (C)AFP; (D) SOX1; (E) ZIC1; and (F) NFM.

FIGS. 37A-C are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence or absence of combinations of retinoic acid(RA), fibroblast growth factor-10 (FGF-10) and fibroblast growthfactor-4 (FGF-4) added on day 4. The panels show the relative levels ofexpression of the following marker genes: (A) PDX1; (B) SOX7; and (C)NFM.

FIGS. 38A-G are charts which show the relative expression of markergenes in a culture of definitive endoderm cells contacted with 50 ng/mlFGF-10 in combination with either 1 μM, 0.2 μM or 0.04 μM retinoic acid(RA) added on day 4. The panels show the relative levels of expressionof the following marker genes: (A) PDX1; (B) HOXA3; (C) HOXC6; (D)HOXA13; (E) CDX1; (F) SOX1; and (G) NFM.

FIGS. 39A-E are charts which show the relative expression of markergenes in a culture of hESCs after 4 days and 8 days with and withoutactivin in the presence of combinations of retinoic acid (RA),fibroblast growth factor-10 (FGF-10) and one of the following: serumreplacement (SR), fetal bovine serum (FBS) or B27. The panels show therelative levels of expression of the following marker genes: (A) PDX1;(B) SOX7; (C) AFP; (D) ZIC1; and (E) NFM.

FIGS. 40A-B are charts which show the relative expression of markergenes for pancreas (PDX1, HNF6) and liver (HNF6) in a culture of hESCsafter 6 days (Oust prior to addition of RA) and at 9 days (three daysafter exposure to RA). Various conditions were included to compare theaddition of activin B at doses of 10 ng/ml (a10), 25 ng/ml (a25) or 50ng/ml (a50) in the presence of either 25 ng/ml (A25) or 50 ng/ml (A50)activin A. The condition without any activin A or activin B (NF) servesas the negative control for definitive endoderm and PDX1-positiveendoderm production. The panels show the relative levels of expressionof the following marker genes: (A) PDX1 and (B) HNF6.

FIGS. 41A-C are charts which show the relative expression of markergenes in a culture of hESCs with 100 ng/ml (A100), 50 ng/ml (A50) orwithout (NF) activin A at 5 days (just prior to retinoic acid addition)and at 2, 4, and 6 days after RA exposure (day 7, 9, and 11,respectively). The percentage label directly under each bar indicatesthe FBS dose during days 3-5 of differentiation. Starting at day 7,cells treated with RA (R) were grown in RPMI medium comprising 0.5% FBS.The RA concentration was 2 μM on day 7, 1 μM on day 9 and 0.2 μM on day11. The panels show the relative levels of expression of the followingmarker genes: (A) PDX1; (B) ZIC1; (C) SOX7.

FIGS. 42A-B are charts which show the relative expression of markergenes in a culture of hESCs treated first with activin A in low FBS toinduce definitive endoderm (day 5) and then with fresh (A25R) mediumcomprising 25 ng/ml activin A and RA or various conditioned media(MEFCM, CM#2, CM#3 and CM#4) and RA to induce PDX1-expressing endoderm.Marker expression was determined on days 5, 6, 7, 8 and 9. The panelsshow the relative levels of expression of the following marker genes:(A) PDX1; (B) CDX1.

FIG. 43 is a chart which shows the relative expression of PDX1 in aculture of hESCs treated first with activin A in low FBS to inducedefinitive endoderm and followed by fresh media comprising activin A andretinoic acid (A25R) or varying amounts of RA in conditioned mediadiluted into fresh media. Total volume of media is 5 ml in all cases.

FIG. 44 is a Western blot showing PDX1 immunoprecipitated fromRA-treated definitive endoderm cells 3 days (d8) and 4 days (d9) afterthe addition of RA and 50 ng/ml activin A.

FIG. 45 is a summary chart displaying the results of afluorescence-activated cell sort (FACs) of PDX1-positive foregutendoderm cells genetically tagged with a EGFP reporter under control ofthe PDX1 promoter.

FIG. 46 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg) and EGFP-positive cells (GFP+).

FIG. 47 is a chart showing relative PDX1 expression levels normalized tohousekeeping genes for sorted populations of live cells (Live),EGFP-negative cells (Neg), the half of the EGFP-positive cell populationthat has the lowest EGFP signal intensity (Lo) and the half of theEGFP-positive cell population that has the highest EGFP signal intensity(Hi).

FIGS. 48A-E are a charts showing the relative expression levelsnormalized to housekeeping genes of five pancreatic endoderm markers insorted populations of live cells (Live), EGFP-negative cells (Neg) andEGFP-positive cells (GFP+). Panels: A—NKX2.2; B—GLUT2; C—HNF3P; D—KRT19and E—HNF4a.

FIG. 49 are a charts showing the relative expression levels normalizedto housekeeping genes of two non-pancreatic endoderm markers in sortedpopulations of live cells (Live), EGFP-negative cells (Neg) andEGFP-positive cells (GFP+). Panels: A—ZIC1 and B—GFAP.

FIGS. 50A-D show the in vivo differentiation of definitive endodermcells that are transplanted under the kidney capsule ofimmunocompromised mice. Panels: A—hetatoxylin-eosin staining showinggut-tube-like structures; B—antibody immunoreactivity against hepatocytespecific antigen (liver); C—antibody immunoreactivity against villin(intestine); and D—antibody immunoreactivity against CDX2 (intestine).

FIGS. 51 A-C are charts showing the normalized relative expressionlevels of markers for liver (albumin and PROX1) and lung TITF1) tissuesin cells contacted with Wnt3B at 20 ng/ml, FGF2 at 5 ng/ml or FGF2 at100 ng/ml on days 5-10. DE refers to definitive endoderm. Panels:A—albumin, B—PROX1, and C—TITF 1.

FIGS. 52A-L are charts showing the normalized relative expression levelsof markers for liver (AFP, AAT, hHEX, GLUT2, APOA1 and VCAM1) and lung(VWF and CXR4) tissues in cells contacted with Wnt3A at 20-50 ng/ml,FGF2 at 5 ng/ml or FGF2 at 100 ng/ml on days 5-10 and BMP4 on days 9 and10. DE refers to definitive endoderm. Panels: A—AFP, B—AAT, C—GLUKO,D—hHEX, E—TAT, F—hNF4a, G—CYP7A, H—GLUT2, I—APOA1, J—VCAM1, K—VWF, andL—CXCR4.

DETAILED DESCRIPTION

A crucial stage in early human development termed gastrulation occurs2-3 weeks after fertilization. Gastrulation is extremely significantbecause it is at this time that the three primary germ layers are firstspecified and organized (Lu et al., 2001; Schoenwolf and Smith, 2000).The ectoderm is responsible for the eventual formation of the outercoverings of the body and the entire nervous system whereas the heart,blood, bone, skeletal muscle and other connective tissues are derivedfrom the mesoderm. Definitive endoderm is defined as the germ layer thatis responsible for formation of the entire gut tube which includes theesophagus, stomach and small and large intestines, and the organs whichderive from the gut tube such as the lungs, liver, thymus, parathyroidand thyroid glands, gall bladder and pancreas (Grapin-Botton and Melton,2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells andMelton, 1999; Wells and Melton, 2000). A very important distinctionshould be made between the definitive endoderm and the completelyseparate lineage of cells termed primitive endoderm. The primitiveendoderm is primarily responsible for formation of extra-embryonictissues, mainly the parietal and visceral endoderm portions of theplacental yolk sac and the extracellular matrix material of Reichert'smembrane.

During gastrulation, the process of definitive endoderm formation beginswith a cellular migration event in which mesendoderm cells (cellscompetent to form mesoderm or endoderm) migrate through a structurecalled the primitive streak. Definitive endoderm is derived from cells,which migrate through the anterior portion of the streak and through thenode (a specialized structure at the anterior-most region of thestreak). As migration occurs, definitive endoderm populates first themost anterior gut tube and culminates with the formation of theposterior end of the gut tube.

Definitive endoderm and endoderm cells derived therefrom representimportant multipotent starting points for the derivation of cells whichmake up terminally differentiated tissues and/or organs derived from thedefinitive endoderm lineage. Such cells, tissues and/or organs areextremely useful in cell therapies. As such, the methods describedherein for identifying differentiation factors capable of causing thedifferentiation of definitive endoderm cells and/or PDX1 expressingendoderm cells to other cells types derived from the definitive endodermcell lineage are beneficial for the advancement of cell therapy.

In particular, some embodiments of the present invention relate tomethods of identifying one or more differentiation factors that areuseful for differentiating cells in a cell population comprisingPDX1-positive endoderm cells and/or definitive endoderm cells into cellsthat are capable of promoting the differentiation of definitive endodermcells into cells which are precursors for tissues and/or organs whichinclude, but are not limited to, pancreas, liver, lungs, stomach,intestine, thyroid, thymus, pharynx, gallbladder and urinary bladder.

Additional aspects which relate to compositions of definitive endodermcells, PDX1-positive endoderm as well as methods and compositions usefulfor producing such cells are also described herein.

Definitions

Certain terms and phrases as used throughout this application have themeanings provided as follows:

As used herein, “embryonic” refers to a range of developmental stages ofan organism beginning with a single zygote and ending with amulticellular structure that no longer comprises pluripotent ortotipotent cells other than developed gametic cells. In addition toembryos derived by gamete fusion, the term “embryonic” refers to embryosderived by somatic cell nuclear transfer.

As used herein, “multipotent” or “multipotent cell” refers to a celltype that can give rise to a limited number of other particular celltypes.

As used herein, “expression” refers to the production of a material orsubstance as well as the level or amount of production of a material orsubstance. Thus, determining the expression of a specific marker refersto detecting either the relative or absolute amount of the marker thatis expressed or simply detecting the presence or absence of the marker.

As used herein, “marker” refers to any molecule that can be observed ordetected. For example, a marker can include, but is not limited to, anucleic acid, such as a transcript of a specific gene, a polypeptideproduct of a gene, a non-gene product polypeptide, a glycoprotein, acarbohydrate, a glycolipd, a lipid, a lipoprotein or a small molecule(for example, molecules having a molecular weight of less than 10,000amu)

When used in connection with cell cultures and/or cell populations, theterm “portion” means any non-zero amount of the cell culture or cellpopulation, which ranges from a single cell to the entirety of the cellculture or cells population.

With respect to cells in cell cultures or in cell populations, thephrase “substantially free of” means that the specified cell type ofwhich the cell culture or cell population is free, is present in anamount of less than about 5% of the total number of cells present in thecell culture or cell population.

As used herein, “retinoid” refers to retinol, retinal or retinoic acidas well as derivatives of any of these compounds.

By “conditioned medium” is meant, a medium that is altered as comparedto a base medium.

As used herein, “foregut/midgut” refers to cells of the anterior portionof the gut tube as well as cells of the middle portion of the gut tube,including cells of the foregut/midgut junction.

Definitive Endoderm Cells and Processes Related Thereto

Embodiments described herein relate to novel, defined processes for theproduction of definitive endoderm cells in culture by differentiatingpluripotent cells, such as stem cells into multipotent definitiveendoderm cells. As described above, definitive endoderm cells do notdifferentiate into tissues produced from ectoderm or mesoderm, butrather, differentiate into the gut tube as well as organs that arederived from the gut tube. In certain preferred embodiments, thedefinitive endoderm cells are derived from hESCs. Such processes canprovide the basis for efficient production of human endodermal derivedtissues such as pancreas, liver, lung, stomach, intestine, thyroid andthymus. For example, production of definitive endoderm may be the firststep in differentiation of a stem cell to a functional insulin-producingβ-cell. To obtain useful quantities of insulin-producing β-cells, highefficiency of differentiation is desirable for each of thedifferentiation steps that occur prior to reaching the pancreaticislet/β-cell fate. Since differentiation of stem cells to definitiveendoderm cells represents perhaps the earliest step towards theproduction of functional pancreatic islet/β-cells (as shown in FIG. 1),high efficiency of differentiation at this step is particularlydesirable.

In view of the desirability of efficient differentiation of pluripotentcells to definitive endoderm cells, some aspects of the differentiationprocesses described herein relate to in vitro methodology that resultsin approximately 50-80% conversion of pluripotent cells to definitiveendoderm cells. Typically, such methods encompass the application ofculture and growth factor conditions in a defined and temporallyspecified fashion. Further enrichment of the cell population fordefinitive endoderm cells can be achieved by isolation and/orpurification of the definitive endoderm cells from other cells in thepopulation by using a reagent that specifically binds to definitiveendoderm cells. As such, some embodiments described herein relate todefinitive endoderm cells as well as methods for producing and isolatingand/or purifying such cells.

In order to determine the amount of definitive endoderm cells in a cellculture or cell population, a method of distinguishing this cell typefrom the other cells in the culture or in the population is desirable.Accordingly, certain embodiments described herein relate to cell markerswhose presence, absence and/or relative expression levels are specificfor definitive endoderm and methods for detecting and determining theexpression of such markers.

In some embodiments described herein, the presence, absence and/or levelof expression of a marker is determined by quantitative PCR (Q-PCR). Forexample, the amount of transcript produced by certain genetic markers,such as SOX17, CXCR4, OCT4, AFP, TM, SPARC, SOX7, MIXL1, GATA4, HNF3b,GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, CRIP1 and other markers describedherein is determined by quantitative Q-PCR. In other embodiments,immunohistochemistry is used to detect the proteins expressed by theabove-mentioned genes. In still other embodiments, Q-PCR andimmunohistochemical techniques are both used to identify and determinethe amount or relative proportions of such markers.

By using methods, such as those described above, to determine theexpression of one or more appropriate markers, it is possible toidentify definitive endoderm cells, as well as determine the proportionof definitive endoderm cells in a cell culture or cell population. Forexample, in some embodiments of the present invention, the definitiveendoderm cells or cell populations that are produced express the SOX17and/or the CXCR4 gene at a level of about 2 orders of magnitude greaterthan non-definitive endoderm cell types or cell populations. In otherembodiments, the definitive endoderm cells or cell populations that areproduced express the SOX17 and/or the CXCR4 gene at a level of more than2 orders of magnitude greater than non-definitive endoderm cell types orcell populations. In still other embodiments, the definitive endodermcells or cell populations that are produced express one or more of themarkers selected from the group consisting of SOX17, CXCR4, GSC, FGF17,VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 at a level of about 2 or more than 2orders of magnitude greater than non-definitive endoderm cell types orcell populations. In some embodiments described herein, definitiveendoderm cells do not substantially express PDX1.

Embodiments described herein also relate to definitive endodermcompositions. For example, some embodiments relate to cell culturescomprising definitive endoderm, whereas others relate to cellpopulations enriched in definitive endoderm cells. Some preferredembodiments relate to cell cultures which comprise definitive endodermcells, wherein at least about 50-80% of the cells in culture aredefinitive endoderm cells. An especially preferred embodiment relates tocells cultures comprising human cells, wherein at least about 50-80% ofthe human cells in culture are definitive endoderm cells. Because theefficiency of the differentiation procedure can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In other preferred embodiments, conversion of a pluripotentcell population, such as a stem cell population, to substantially puredefinitive endoderm cell population is contemplated.

The compositions and methods described herein have several usefulfeatures. For example, the cell cultures and cell populations comprisingdefinitive endoderm as well as the methods for producing such cellcultures and cell populations are useful for modeling the early stagesof human development. Furthermore, the compositions and methodsdescribed herein can also serve for therapeutic intervention in diseasestates, such as diabetes mellitus. For example, since definitiveendoderm serves as the source for only a limited number of tissues, itcan be used in the development of pure tissue or cell types.

Production of Definitive Endoderm from Pluripotent Cells

Processes for differentiating pluripotent cells to produce cell culturesand enriched cell populations comprising definitive endoderm isdescribed below and in U.S. Pat. No. 11/021,618, entitled DEFINITIVEENDODERM, filed Dec. 23, 2004, the disclosure of which is incorporatedherein by reference in its entirety. In some of these processes, thepluripotent cells used as starting material are stem cells. In certainprocesses, definitive endoderm cell cultures and enriched cellpopulations comprising definitive endoderm cells are produced fromembryonic stem cells. A preferred method for deriving definitiveendoderm cells utilizes human embryonic stem cells as the startingmaterial for definitive endoderm production. Such pluripotent cells canbe cells that originate from the morula, embryonic inner cell mass orthose obtained from embryonic gonadal ridges. Human embryonic stem cellscan be maintained in culture in a pluripotent state without substantialdifferentiation using methods that are known in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,453,357, 5,670,372,5,690,926 5,843,780, 6,200,806 and 6,251,671 the disclosures of whichare incorporated herein by reference in their entireties.

In some processes for producing definitive endoderm cells, hESCs aremaintained on a feeder layer. In such processes, any feeder layer whichallows hESCs to be maintained in a pluripotent state can be used. Onecommonly used feeder layer for the cultivation of human embryonic stemcells is a layer of mouse fibroblasts. More recently, human fibroblastfeeder layers have been developed for use in the cultivation of hESCs(see U.S. Patent Application No. 2002/0072117, the disclosure of whichis incorporated herein by reference in its entirety). Alternativeprocesses for producing definitive endoderm permit the maintenance ofpluripotent hESC without the use of a feeder layer. Methods ofmaintaining pluripotent hESCs under feeder-free conditions have beendescribed in U.S. Patent Application No. 2003/0175956, the disclosure ofwhich is incorporated herein by reference in its entirety.

The human embryonic stem cells used herein can be maintained in cultureeither with or without serum. In some embryonic stem cell maintenanceprocedures, serum replacement is used. In others, serum free culturetechniques, such as those described in U.S. Patent Application No.2003/0190748, the disclosure of which is incorporated herein byreference in its entirety, are used.

Stem cells are maintained in culture in a pluripotent state by routinepassage until it is desired that they be differentiated into definitiveendoderm. In some processes, differentiation to definitive endoderm isachieved by providing to the stem cell culture a growth factor of theTGFβ superfamily in an amount sufficient to promote differentiation todefinitive endoderm. Growth factors of the TGFβ superfamily which areuseful for the production of definitive endoderm are selected from theNodal/Activin or BMP subgroups. In some preferred differentiationprocesses, the growth factor is selected from the group consisting ofNodal, activin A, activin B and BMP4. Additionally, the growth factorWnt3a and other Wnt family members are useful for the production ofdefinitive endoderm cells. In certain differentiation processes,combinations of any of the above-mentioned growth factors can be used.

With respect to some of the processes for the differentiation ofpluripotent stem cells to definitive endoderm cells, the above-mentionedgrowth factors are provided to the cells so that the growth factors arepresent in the cultures at concentrations sufficient to promotedifferentiation of at least a portion of the stem cells to definitiveendoderm cells. In some processes, the above-mentioned growth factorsare present in the cell culture at a concentration of at least about 5ng/ml, at least about 10 ng/ml, at least about 25 ng/ml, at least about50 ng/ml, at least about 75 ng/ml, at least about 100 ng/ml, at leastabout 200 ng/ml, at least about 300 ng/ml, at least about 400 ng/ml, atleast about 500 ng/ml, at least about 1000 ng/ml, at least about 2000ng/ml, at least about 3000 ng/ml, at least about 4000 ng/ml, at leastabout 5000 ng/ml or more than about 5000 ng/ml.

In certain processes for the differentiation of pluripotent stem cellsto definitive endoderm cells, the above-mentioned growth factors areremoved from the cell culture subsequent to their addition. For example,the growth factors can be removed within about one day, about two days,about three days, about four days, about five days, about six days,about seven days, about eight days, about nine days or about ten daysafter their addition. In a preferred processes, the growth factors areremoved about four days after their addition.

Cultures of definitive endoderm cells can be grown in medium containingreduced serum or no serum. Under certain culture conditions, serumconcentrations can range from about 0.05% v/v to about 20% v/v. Forexample, in some differentiation processes, the serum concentration ofthe medium can be less than about 0.05% (v/v), less than about 0.1%(v/v), less than about 0.2% (v/v), less than about 0.3% (v/v), less thanabout 0.4% (v/v), less than about 0.5% (v/v), less than about 0.6%(v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less thanabout 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v),less than about 3% (v/v), less than about 4% (v/v), less than about 5%(v/v), less than about 6% (v/v), less than about 7% (v/v), less thanabout 8% (v/v), less than about 9% (v/v), less than about 10% (v/v),less than about 15% (v/v) or less than about 20% (v/v). In someprocesses, definitive endoderm cells are grown without serum or withserum replacement. In still other processes, definitive endoderm cellsare grown in the presence of B27. In such processes, the concentrationof B27 supplement can range from about 0.1% v/v to about 20% v/v.

Monitoring the Differentiation of Pluripotent Cells to DefinitiveEndoderm

The progression of the HESC culture to definitive endoderm can bemonitored by determining the expression of markers characteristic ofdefinitive endoderm. In some processes, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such processes, themeasurement of marker expression can be qualitative or quantitative. Onemethod of quantitating the expression of markers that are produced bymarker genes is through the use of quantitative PCR (Q-PCR). Methods ofperforming Q-PCR are well known in the art. Other methods which areknown in the art can also be used to quantitate marker gene expression.For example, the expression of a marker gene product can be detected byusing antibodies specific for the marker gene product of interest. Incertain processes, the expression of marker genes characteristic ofdefinitive endoderm as well as the lack of significant expression ofmarker genes characteristic of hESCs and other cell types is determined.

As described further in the Examples below, a reliable marker ofdefinitive endoderm is the SOX17 gene. As such, the definitive endodermcells produced by the processes described herein express the SOX17marker gene, thereby producing the SOX17 gene product. Other markers ofdefinitive endoderm are MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP1. Since definitive endoderm cells express theSOX17 marker gene at a level higher than that of the SOX7 marker gene,which is characteristic of primitive and visceral endoderm (see Table1), in some processes, the expression of both SOX17 and SOX7 ismonitored. In other processes, expression of the both the SOX17 markergene and the OCT4 marker gene, which is characteristic of hESCs, ismonitored. Additionally, because definitive endoderm cells express theSOX17 marker gene at a level higher than that of the AFP, SPARC orThrombomodulin (TM) marker genes, the expression of these genes can alsobe monitored.

Another marker of definitive endoderm is the CXCR4 gene. The CXCR4 geneencodes a cell surface chemokine receptor whose ligand is thechemoattractant SDF-1. The principal roles of the CXCR4 receptor-bearingcells in the adult are believed to be the migration of hematopoeticcells to the bone marrow, lymphocyte trafficking and the differentiationof various B cell and macrophage blood cell lineages [Kim, C., andBroxmeyer, H. J. Leukocyte Biol. 65, 6-15 (1999)]. The CXCR4 receptoralso functions as a coreceptor for the entry of HIV-1 into T-cells[Feng, Y., et al. Science, 272, 872-877 (1996)]. In an extensive seriesof studies carried out by [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)], the expression of the chemokine receptor CXCR4 and itsunique ligand, SDF-1 [Kim, C., and Broxmyer, H., J. Leukocyte Biol. 65,6-15 (1999)], were delineated during early development and adult life inthe mouse. The CXCR4/SDF1 interaction in development became apparentwhen it was demonstrated that if either gene was disrupted in transgenicmice [Nagasawa et al. Nature, 382, 635-638 (1996)], Ma, Q., et alImmunity, 10, 463-471 (1999)] it resulted in late embryonic lethality.McGrath et al. demonstrated that CXCR4 is the most abundant chemokinereceptor messenger RNA detected during early gastrulating embryos (E7.5)using a combination of RNase protection and in situ hybridizationmethodologies. In the gastrulating embryo, CXCR4/SDF-1 signaling appearsto be mainly involved in inducing migration of primitive-streakgermlayer cells and is expressed on definitive endoderm, mesoderm andextraembryonic mesoderm present at this time. In E7.2-7.8 mouse embryos,CXCR4 and alpha-fetoprotein are mutually exclusive indicating a lack ofexpression in visceral endoderm [McGrath, K. E. et al. Dev. Biology 213,442-456 (1999)].

Since definitive endoderm cells produced by differentiating pluripotentcells express the CXCR4 marker gene, expression of CXCR4 can bemonitored in order to track the production of definitive endoderm cells.Additionally, definitive endoderm cells produced by the methodsdescribed herein express other markers of definitive endoderm including,but not limited to, SOX17, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR,FOXQ1, CMKOR1 and CRIP 1. Since definitive endoderm cells express theCXCR4 marker gene at a level higher than that of the SOX7 marker gene,the expression of both CXCR4 and SOX7 can be monitored. In otherprocesses, expression of both the CXCR4 marker gene and the OCT4 markergene, is monitored. Additionally, because definitive endoderm cellsexpress the CXCR4 marker gene at a level higher than that of the AFP,SPARC or Thrombomodulin (TM) marker genes, the expression of these genescan also be monitored.

It will be appreciated that expression of CXCR4 in endodermal cells doesnot preclude the expression of SOX17. As such, definitive endoderm cellsproduced by the processes described herein will substantially expressSOX17 and CXCR4 but will not substantially express AFP, TM, SPARC orPDX1.

It will be appreciated that SOX17 and/or CXCR4 marker expression isinduced over a range of different levels in definitive endoderm cellsdepending on the differentiation conditions. As such, in someembodiments described herein, the expression of the SOX17 marker and/orthe CXCR4 marker in definitive endoderm cells or cell populations is atleast about 2-fold higher to at least about 10,000-fold higher than theexpression of the SOX17 marker and/or the CXCR4 marker in non-definitiveendoderm cells or cell populations, for example pluripotent stem cells.In other embodiments, the expression of the SOX17 marker and/or theCXCR4 marker in definitive endoderm cells or cell populations is atleast about 4-fold higher, at least about 6-fold higher, at least about8-fold higher, at least about 10-fold higher, at least about 15-foldhigher, at least about 20-fold higher, at least about 40-fold higher, atleast about 80-fold higher, at least about 100-fold higher, at leastabout 150-fold higher, at least about 200-fold higher, at least about500-fold higher, at least about 750-fold higher, at least about1000-fold higher, at least about 2500-fold higher, at least about5000-fold higher, at least about 7500-fold higher or at least about10,000-fold higher than the expression of the SOX17 marker and/or theCXCR4 marker in non-definitive endoderm cells or cell populations, forexample pluripotent stem cells. In some embodiments, the expression ofthe SOX17 marker and/or CXCR4 marker in definitive endoderm cells orcell populations is infinitely higher than the expression of the SOX17marker and/or the CXCR4 marker in non-definitive endoderm cells or cellpopulations, for example pluripotent stem cells.

It will also be appreciated that in some embodiments described herein,the expression of markers selected from the group consisting of GATA4,MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 indefinitive endoderm cells or cell populations is increased as comparedto the expression of GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1 in non-definitive endoderm cells or cell populations.

Additionally, it will be appreciated that there is a range ofdifferences between the expression level of the SOX17 marker and theexpression levels of the OCT4, SPARC, AFP, TM and/or SOX7 markers indefinitive endoderm cells. Similarly, there exists a range ofdifferences between the expression level of the CXCR4 marker and theexpression levels of the OCT4, SPARC, AFP, TM and/or SOX7 markers indefinitive endoderm cells. As such, in some embodiments describedherein, the expression of the SOX17 marker or the CXCR4 marker is atleast about 2-fold higher to at least about 10,000-fold higher than theexpression of OCT4, SPARC, AFP, TM and/or SOX7 markers. In otherembodiments, the expression of the SOX17 marker or the CXCR4 marker isat least about 4-fold higher, at least about 6-fold higher, at leastabout 8-fold higher, at least about 10-fold higher, at least about15-fold higher, at least about 20-fold higher, at least about 40-foldhigher, at least about 80-fold higher, at least about 100-fold higher,at least about 150-fold higher, at least about 200-fold higher, at leastabout 500-fold higher, at least about 750-fold higher, at least about1000-fold higher, at least about 2500-fold higher, at least about5000-fold higher, at least about 7500-fold higher or at least about10,000-fold higher than the expression of OCT4, SPARC, AFP, TM and/orSOX7 markers. In some embodiments, OCT4, SPARC, AFP, TM and/or SOX7markers are not significantly expressed in definitive endoderm cells.

It will also be appreciated that in some embodiments described herein,the expression of markers selected from the group consisting of GATA4,MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 indefinitive endoderm cells is increased as compared to the expression ofOCT4, SPARC, AFP, TM and/or SOX7 in definitive endoderm cells.

Enrichment, Isolation and/or Purification of Definitive Endoderm

Definitive endoderm cells produced by any of the above-describedprocesses can be enriched, isolated and/or purified by using an affinitytag that is specific for such cells. Examples of affinity tags specificfor definitive endoderm cells are antibodies, ligands or other bindingagents that are specific to a marker molecule, such as a polypeptide,that is present on the cell surface of definitive endoderm cells butwhich is not substantially present on other cell types that would befound in a cell culture produced by the methods described herein. Insome processes, an antibody which binds to CXCR4 is used as an affinitytag for the enrichment, isolation or purification of definitive endodermcells. In other processes, the chemokine SDF-1 or other molecules basedon SDF-1 can also be used as affinity tags. Such molecules include, butnot limited to, SDF-1 fragments, SDF-1 fusions or SDF-1 mimetics.

Methods for making antibodies and using them for cell isolation areknown in the art and such methods can be implemented for use with theantibodies and definitive endoderm cells described herein. In oneprocess, an antibody which binds to CXCR4 is attached to a magnetic beadand then allowed to bind to definitive endoderm cells in a cell culturewhich has been enzymatically treated to reduce intercellular andsubstrate adhesion. The cell/antibody/bead complexes are then exposed toa movable magnetic field which is used to separate bead-bound definitiveendoderm cells from unbound cells. Once the definitive endoderm cellsare physically separated from other cells in culture, the antibodybinding is disrupted and the cells are replated in appropriate tissueculture medium.

Additional methods for obtaining enriched, isolated or purifieddefinitive endoderm cell cultures or populations can also be used. Forexample, in some embodiments, the CXCR4 antibody is incubated with adefinitive endoderm-containing cell culture that has been treated toreduce intercellular and substrate adhesion. The cells are then washed,centrifuged and resuspended. The cell suspension is then incubated witha secondary antibody, such as an FITC-conjugated antibody that iscapable of binding to the primary antibody. The cells are then washed,centrifuged and resuspended in buffer. The cell suspension is thenanalyzed and sorted using a fluorescence activated cell sorter (FACS).CXCR4-positive cells are collected separately from CXCR4-negative cells,thereby resulting in the isolation of such cell types. If desired, theisolated cell compositions can be further purified by using an alternateaffinity-based method or by additional rounds of sorting using the sameor different markers that are specific for definitive endoderm.

In still other processes, definitive endoderm cells are enriched,isolated and/or purified using a ligand or other molecule that binds toCXCR4. In some processes, the molecule is SDF-1 or a fragment, fusion ormimetic thereof.

In preferred processes, definitive endoderm cells are enriched, isolatedand/or purified from other non-definitive endoderm cells after the stemcell cultures are induced to differentiate towards the definitiveendoderm lineage. It will be appreciated that the above-describedenrichment, isolation and purification procedures can be used with suchcultures at any stage of differentiation.

In addition to the procedures just described, definitive endoderm cellsmay also be isolated by other techniques for cell isolation.Additionally, definitive endoderm cells may also be enriched or isolatedby methods of serial subculture in growth conditions which promote theselective survival or selective expansion of the definitive endodermcells.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of definitive endoderm cells and or tissues can be producedin vitro from pluripotent cell cultures or cell populations, such asstem cell cultures or populations, which have undergone at least somedifferentiation. In some methods, the cells undergo randomdifferentiation. In a preferred method, however, the cells are directedto differentiate primarily into definitive endoderm. Some preferredenrichment, isolation and/or purification methods relate to the in vitroproduction of definitive endoderm from human embryonic stem cells. Usingthe methods described herein, cell populations or cell cultures can beenriched in definitive endoderm content by at least about 2- to about1000-fold as compared to untreated cell populations or cell cultures. Insome embodiments, definitive endoderm cells can be enriched by at leastabout 5- to about 500-fold as compared to untreated cell populations orcell cultures. In other embodiments, definitive endoderm cells can beenriched from at least about 10- to about 200-fold as compared tountreated cell populations or cell cultures. In still other embodiments,definitive endoderm cells can be enriched from at least about 20- toabout 100-fold as compared to untreated cell populations or cellcultures. In yet other embodiments, definitive endoderm cells can beenriched from at least about 40- to about 80-fold as compared tountreated cell populations or cell cultures. In certain embodiments,definitive endoderm cells can be enriched from at least about 2- toabout 20-fold as compared to untreated cell populations or cellcultures.

Compositions Comprising Definitive Endoderm

Cell compositions produced by the above-described methods include cellcultures comprising definitive endoderm and cell populations enriched indefinitive endoderm. For example, cell cultures which comprisedefinitive endoderm cells, wherein at least about 50-80% of the cells inculture are definitive endoderm cells, can be produced. Because theefficiency of the differentiation process can be adjusted by modifyingcertain parameters, which include but are not limited to, cell growthconditions, growth factor concentrations and the timing of culturesteps, the differentiation procedures described herein can result inabout 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, orgreater than about 95% conversion of pluripotent cells to definitiveendoderm. In processes in which isolation of definitive endoderm cellsis employed, for example, by using an affinity reagent that binds to theCXCR4 receptor, a substantially pure definitive endoderm cell populationcan be recovered.

Some embodiments described herein relate to compositions, such as cellpopulations and cell cultures, that comprise both pluripotent cells,such as stem cells, and definitive endoderm cells. For example, usingthe methods described herein, compositions comprising mixtures of hESCsand definitive endoderm cells can be produced. In some embodiments,compositions comprising at least about 5 definitive endoderm cells forabout every 95 pluripotent cells are produced. In other embodiments,compositions comprising at least about 95 definitive endoderm cells forabout every 5 pluripotent cells are produced. Additionally, compositionscomprising other ratios of definitive endoderm cells to pluripotentcells are contemplated. For example, compositions comprising at leastabout 1 definitive endoderm cell for about every 1,000,000 pluripotentcells, at least about 1 definitive endoderm cell for about every 100,000pluripotent cells, at least about 1 definitive endoderm cell for aboutevery 10,000 pluripotent cells, at least about 1 definitive endodermcell for about every 1000 pluripotent cells, at least about 1 definitiveendoderm cell for about every 500 pluripotent cells, at least about 1definitive endoderm cell for about every 100 pluripotent cells, at leastabout 1 definitive endoderm cell for about every 10 pluripotent cells,at least about 1 definitive endoderm cell for about every 5 pluripotentcells, at least about 1 definitive endoderm cell for about every 2pluripotent cells, at least about 2 definitive endoderm cells for aboutevery 1 pluripotent cell, at least about 5 definitive endoderm cells forabout every 1 pluripotent cell, at least about 10 definitive endodermcells for about every 1 pluripotent cell, at least about 20 definitiveendoderm cells for about every 1 pluripotent cell, at least about 50definitive endoderm cells for about every 1 pluripotent cell, at leastabout 100 definitive endoderm cells for about every 1 pluripotent cell,at least about 1000 definitive endoderm cells for about every 1pluripotent cell, at least about 10,000 definitive endoderm cells forabout every 1 pluripotent cell, at least about 100,000 definitiveendoderm cells for about every 1 pluripotent cell and at least about1,000,000 definitive endoderm cells for about every 1 pluripotent cellare contemplated. In some embodiments, the pluripotent cells are humanpluripotent stem cells. In certain embodiments the stem cells arederived from a morula, the inner cell mass of an embryo or the gonadalridges of an embryo. In certain other embodiments, the pluripotent cellsare derived from the gondal or germ tissues of a multicellular structurethat has developed past the embryonic stage.

Some embodiments described herein relate to cell cultures or cellpopulations comprising from at least about 5% definitive endoderm cellsto at least about 95% definitive endoderm cells. In some embodiments thecell cultures or cell populations comprise mammalian cells. In preferredembodiments, the cell cultures or cell populations comprise human cells.For example, certain specific embodiments relate to cell culturescomprising human cells, wherein from at least about 5% to at least about95% of the human cells are definitive endoderm cells. Other embodimentsrelate to cell cultures comprising human cells, wherein at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90% or greaterthan 90% of the human cells are definitive endoderm cells. Inembodiments where the cell cultures or cell populations comprise humanfeeder cells, the above percentages are calculated without respect tothe human feeder cells in the cell cultures or cell populations.

Further embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising human cells, such as humandefinitive endoderm cells, wherein the expression of either the SOX17 orthe CXCR4 marker is greater than the expression of the OCT4, SPARC,alpha-fetoprotein (AFP), Thrombomodulin (TM) and/or SOX7 marker in atleast about 5% of the human cells. In other embodiments, the expressionof either the SOX17 or the CXCR4 marker is greater than the expressionof the OCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 10% ofthe human cells, in at least about 15% of the human cells, in at leastabout 20% of the human cells, in at least about 25% of the human cells,in at least about 30% of the human cells, in at least about 35% of thehuman cells, in at least about 40% of the human cells, in at least about45% of the human cells, in at least about 50% of the human cells, in atleast about 55% of the human cells, in at least about 60% of the humancells, in at least about 65% of the human cells, in at least about 70%of the human cells, in at least about 75% of the human cells, in atleast about 80% of the human cells, in at least about 85% of the humancells, in at least about 90% of the human cells, in at least about 95%of the human cells or in greater than 95% of the human cells. Inembodiments where the cell cultures or cell populations comprise humanfeeder cells, the above percentages are calculated without respect tothe human feeder cells in the cell cultures or cell populations.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations, comprisinghuman cells, such as human definitive endoderm cells, wherein theexpression of one or more markers selected from the group consisting ofGATA4, MIXLI, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7markers in from at least about 5% to greater than at least about 95% ofthe human cells. In embodiments where the cell cultures or cellpopulations comprise human feeder cells, the above percentages arecalculated without respect to the human feeder cells in the cellcultures or cell populations.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising human cells, such as humandefinitive endoderm cells, wherein the expression both the SOX17 and theCXCR4 marker is greater than the expression of the OCT4, SPARC, AFP, TMand/or SOX7 marker in at least about 5% of the human cells. In otherembodiments, the expression of both the SOX17 and the CXCR4 marker isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7marker in at least about 10% of the human cells, in at least about 15%of the human cells, in at least about 20% of the human cells, in atleast about 25% of the human cells, in at least about 30% of the humancells, in at least about 35% of the human cells, in at least about 40%of the human cells, in at least about 45% of the human cells, in atleast about 50% of the human cells, in at least about 55% of the humancells, in at least about 60% of the human cells, in at least about 65%of the human cells, in at least about 70% of the human cells, in atleast about 75% of the human cells, in at least about 80% of the humancells, in at least about 85% of the human cells, in at least about 90%of the human cells, in at least about 95% of the human cells or ingreater than 95% of the human cells. In embodiments where the cellcultures or cell populations comprise human feeder cells, the abovepercentages are calculated without respect to the human feeder cells inthe cell cultures or cell populations.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations, comprisinghuman cells, such as human definitive endoderm cells, wherein theexpression of the GATA4, MIXL1, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1 markers is greater than the expression of the OCT4,SPARC, AFP, TM and/or SOX7 markers in from at least about 5% to greaterthan at least about 95% of the human cells. In embodiments where thecell cultures or cell populations comprise human feeder cells, the abovepercentages are calculated without respect to the human feeder cells inthe cell cultures or cell populations.

Additional embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endoderm cells, wherein the expression of eitherthe SOX17 or the CXCR4 marker is greater than the expression of theOCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 5% of theendodermal cells. In other embodiments, the expression of either theSOX17 or the CXCR4 marker is greater than the expression of the OCT4,SPARC, AFP, TM and/or SOX7 marker in at least about 10% of theendodermal cells, in at least about 15% of the endodermal cells, in atleast about 20% of the endodermal cells, in at least about 25% of theendodermal cells, in at least about 30% of the endodermal cells, in atleast about 35% of the endodermal cells, in at least about 40% of theendodermal cells, in at least about 45% of the endodermal cells, in atleast about 50% of the endodermal cells, in at least about 55% of theendodermal cells, in at least about 60% of the endodermal cells, in atleast about 65% of the endodermal cells, in at least about 70% of theendodermal cells, in at least about 75% of the endodermal cells, in atleast about 80% of the endodermal cells, in at least about 85% of theendodermal cells, in at least about 90% of the endodermal cells, in atleast about 95% of the endodermal cells or in greater than 95% of theendodermal cells.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations comprisingmammalian endodermal cells, wherein the expression of one or moremarkers selected from the group consisting of GATA4, MIXL1, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 is greater than theexpression of the OCT4, SPARC, AFP, TM and/or SOX7 markers in from atleast about 5% to greater than at least about 95% of the endodermalcells.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endodermal cells, wherein the expression of boththe SOX17 and the CXCR4 marker is greater than the expression of theOCT4, SPARC, AFP, TM and/or SOX7 marker in at least about 5% of theendodermal cells. In other embodiments, the expression of both the SOX17and the CXCR4 marker is greater than the expression of the OCT4, SPARC,AFP, TM and/or SOX7 marker in at least about 10% of the endodermalcells, in at least about 15% of the endodermal cells, in at least about20% of the endodermal cells, in at least about 25% of the endodermalcells, in at least about 30% of the endodermal cells, in at least about35% of the endodermal cells, in at least about 40% of the endodermalcells, in at least about 45% of the endodermal cells, in at least about50% of the endodermal cells, in at least about 55% of the endodermalcells, in at least about 60% of the endodermal cells, in at least about65% of the endodermal cells, in at least about 70% of the endodermalcells, in at least about 75% of the endodermal cells, in at least about80% of the endodermal cells, in at least about 85% of the endodermalcells, in at least about 90% of the endodermal cells, in at least about95% of the endodermal cells or in greater than 95% of the endodermalcells.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations comprisingmammalian endodermal cells, wherein the expression of the GATA4, MIXL1,HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1 markers isgreater than the expression of the OCT4, SPARC, AFP, TM and/or SOX7markers in from at least about 5% to greater than at least about 95% ofthe endodermal cells.

Using the methods described herein, compositions comprising definitiveendoderm cells substantially free of other cell types can be produced.In some embodiments described herein, the definitive endoderm cellpopulations or cell cultures produced by the methods described hereinare substantially free of cells that significantly express the OCT4,SOX7, AFP, SPARC, TM, ZIC1 or BRACH marker genes.

In one embodiment, a description of a definitive endoderm cell based onthe expression of marker genes is, SOX17 high, MIXL1 high, AFP low,SPARC low, Thrombomodulin low, SOX7 low, CXCR4 high.

The PDX1 Gene Expression During Development

PDX1 (also called STF-1, IDX-1 and IPF-1) is a transcription factor thatis necessary for development of the pancreas and rostral duodenum. PDX1is first expressed in the pancreatic endoderm, which arises fromposterior foregut endoderm and will produce both the exocrine andendocrine cells, starting at E8.5 in the mouse. Later, PDX1 becomesrestricted to beta-cells and some delta-cells of the endocrine pancreas.This expression pattern is maintained in the adult. PDX1 is alsoexpressed in duodenal endoderm early in development, which is adjacentto the forming pancreas, then in the duodenal enterocytes andenteroendocrine cells, antral stomach and in the common bile, cystic andbiliary ducts. This region of expression also becomes limited, at thetime that pancreatic expression becomes restricted, to predominantly therostral duodenum.

PDX1-Positive Cells and Processes Related Thereto

Embodiments of other differentiation processes described herein relateto novel, defined processes for the production of PDX1-positive endodermcells, wherein the PDX1-positive endoderm cells are multipotent cellsthat can differentiate into cells, tissues or organs derived from theforegut/midgut region of the gut tube (PDX1-positive foregut/midgutendoderm). Some preferred embodiments relate to processes for theproduction of PDX1-positive foregut endoderm cells. In some embodiments,these PDX1-positive foregut endoderm cells are multipotent cells thatcan differentiate into cells, tissues or organs derived from theanterior portion of the gut tube (PDX1-positive foregut endoderm).Additional preferred embodiments relate to processes for the productionof PDX1-positive endoderm cells of the posterior portion of the foregut.In some embodiments, these PDX1-positive endoderm cells are multipotentcells that can differentiate into cells, tissues or organs derived fromthe posterior portion of the foregut region of the gut tube.

The PDX1-positive foregut endoderm cells, such as those producedaccording to the methods described herein, can be used to produce fullydifferentiated insulin-producing β-cells. In some embodiments,PDX1-positive foregut endoderm cells are produced by differentiatingdefinitive endoderm cells that do not substantially express PDX1(PDX1-negative definitive endoderm cells; also referred to herein asdefinitive endoderm) so as to form PDX1-positive foregut endoderm cells.PDX1-negative definitive endoderm cells can be prepared bydifferentiating pluripotent cells, such as embryonic stem cells, asdescribed herein or by any other known methods. A convenient and highlyefficient method for producing PDX1-negative definitive endoderm frompluripotent cells is described previously herein and in U.S. Pat. No.11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, thedisclosure of which is incorporated herein by reference in its entirety.

Processes of producing PDX1-positive foregut endoderm cells provide abasis for efficient production of pancreatic tissues such as acinarcells, ductal cells and islet cells from pluripotent cells. In certainpreferred embodiments, human PDX1-positive foregut endoderm cells arederived from human PDX1-negative definitive endoderm cells, which inturn, are derived from hESCs. These human PDX1-positive foregut endodermcells can then be used to produce functional insulin-producing β-cells.To obtain useful quantities of insulin-producing β-cells, highefficiency of differentiation is desirable for each of thedifferentiation steps that occur prior to reaching the pancreaticislet/β-cell fate. Because differentiation of PDX1-negative definitiveendoderm cells to PDX1 -positive foregut endoderm cells represents anearly step towards the production of functional pancreatic islet/β-cells(as shown in FIG. 1), high efficiency of differentiation at this step isparticularly desirable.

In view of the desirability of efficient differentiation ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells, some aspects of the processes described herein relate toin vitro methodology that results in approximately 2-25% conversion ofPDX1-negative definitive endoderm cells to PDX1-positive foregutendoderm cells. Typically, such methods encompass the application ofculture and growth factor conditions in a defined and temporallyspecified fashion. Further enrichment of the cell population forPDX1-positive foregut endoderm cells can be achieved by isolation and/orpurification of the PDX1-positive foregut endoderm cells from othercells in the population by using a reagent that specifically binds tothe PDX1-positive foregut endoderm cells. As an alternative,PDX1-positive foregut endoderm cells can be labeled with a reportergene, such as green fluorescent protein (GFP), so as to enable thedetection of PDX1 expression. Such fluorescently labeled cells can thenbe purified by fluorescent activated cell sorting (FACS). Furtheraspects of the present invention relate to cell cultures and enrichedcell populations comprising PDX1-positive foregut endoderm cells as wellas methods for identifying factors useful in the differentiation to andfrom PDX1-positive foregut endoderm.

In order to determine the amount of PDX1-positive foregut endoderm cellsin a cell culture or cell population, a method of distinguishing thiscell type from the other cells in the culture or in the population isdesirable. Accordingly, certain embodiments described herein relate tocell markers whose presence, absence and/or relative expression levelsare indicative of PDX1-positive foregut endoderm cells as well asmethods for detecting and determining the expression of such markers.

In some embodiments described herein, the presence, absence and/or levelof expression of a marker is determined by quantitative PCR (Q-PCR). Forexample, the amount of transcript produced by certain genetic markers,such as PDX1, SOX17, SOX7, SOX1, ZIC1, NFM, alpha-fetoprotein (AFP),homeobox A13 (HOXA13), homeobox C6 (HOXC6), and/or other markersdescribed herein is determined by Q-PCR. In other embodiments,immunohistochemistry is used to detect the proteins expressed by theabove-mentioned genes. In still other embodiments, Q-PCR andimmunohistochemical techniques are both used to identify and determinethe amount or relative proportions of such markers.

By using the differentiation and detection methods described herein, itis possible to identify PDX1-positive foregut endoderm cells, as well asdetermine the proportion of PDX1-positive foregut endoderm cells in acell culture or cell population. For example, in some embodiments, thePDX1-positive foregut endoderm cells or cell populations that areproduced express the PDX1 gene at a level of at least about 2 orders ofmagnitude greater than PDX1-negative cells or cell populations. In otherembodiments, the PDX1-positive foregut endoderm cells and cellpopulations that are produced express the PDX1 gene at a level of morethan 2 orders of magnitude greater than PDX1-negative cells or cellpopulations. In still other embodiments, the PDX1-positive foregutendoderm cells or cell populations that are produced express one or moreof the markers selected from the group consisting of PDX1, SOX17, HOXA13and HOXC6 at a level of about 2 or more than 2 orders of magnitudegreater than PDX1-negative definitive endoderm cells or cellpopulations.

The compositions and methods described herein have several usefulfeatures. For example, the cell cultures and cell populations comprisingPDX1-positive endoderm, as well as the methods for producing such cellcultures and cell populations, are useful for modeling the early stagesof human development. Furthermore, the compositions and methodsdescribed herein can also serve for therapeutic intervention in diseasestates, such as diabetes mellitus. For example, since PDX1-positiveforegut endoderm serves as the source for only a limited number oftissues, it can be used in the development of pure tissue or cell types.

Production of PDX1-Positve Foregut Endoderm from PDX1-NegativeDefinitive Endoderm

The PDX1-positive foregut endoderm cell cultures and populationscomprising PDX1-positive foregut endoderm cells that are describedherein are produced from PDX1-negative definitive endoderm, which isgenerated from pluripotent cells as described above. A preferred methodutilizes human embryonic stem cells as the starting material. In oneembodiment, hESCs are first converted to PDX1-negative definitiveendoderm cells, which are then converted to PDX1-positive foregutendoderm cells. It will be appreciated, however, that the startingmaterials for the production of PDX1-positive foregut endoderm is notlimited to definitive endoderm cells produced using pluripotent celldifferentiation methods. Rather, any PDX1-negative definitive endodermcells can be used in the methods described herein regardless of theirorigin.

In some embodiments described herein, cell cultures or cell populationscomprising PDX1-negative definitive endoderm cells can be used forfurther differentiation to cell cultures and/or enriched cellpopulations comprising PDX1-positive foregut endoderm cells. Forexample, a cell culture or cell population comprising humanPDX1-negative, SOX17-positive definitive endoderm cells can be used. Insome embodiments, the cell culture or cell population may also comprisedifferentiation factors, such as activins, nodals and/or BMPs, remainingfrom the previous differentiation step (that is, the step ofdifferentiating pluripotent cells to definitive endoderm cells). Inother embodiments, factors utilized in the previous differentiation stepare removed from the cell culture or cell population prior to theaddition of factors used for the differentiation of the PDX1-negative,SOX17-positive definitive endoderm cells to PDX1-positive foregutendoderm cells. In other embodiments, cell populations enriched forPDX1-negative, SOX17-positive definitive endoderm cells are used as asource for the production of PDX1-positive foregut endoderm cells.

PDX1-negative definitive endoderm cells in culture are differentiated toPDX1-positive endoderm cells by providing to a cell culture comprisingPDX1-negative, SOX17-positive definitive endoderm cells adifferentiation factor that promotes differentiation of the cells toPDX1-positive foregut endoderm cells (foregut differentiation factor).In some embodiments of the present invention, the foregutdifferentiation factor is retinoid, such as retinoic acid (RA). In someembodiments, the retinoid is used in conjunction with a fibroblastgrowth factor, such as FGF-4 or FGF-10. In other embodiments, theretinoid is used in conjunction with a member of the TGFβ superfamily ofgrowth factors and/or a conditioned medium.

As defined above, the phrase “conditioned medium” refers to a mediumthat is altered as compared to a base medium. For example, theconditioning of a medium may cause molecules, such as nutrients and/orgrowth factors, to be added to or depleted from the original levelsfound in the base medium. In some embodiments, a medium is conditionedby allowing cells of certain types to be grown or maintained in themedium under certain conditions for a certain period of time. Forexample, a medium can be conditioned by allowing hESCs to be expanded,differentiated or maintained in a medium of defined composition at adefined temperature for a defined number of hours. As will beappreciated by those of skill in the art, numerous combinations ofcells, media types, durations and environmental conditions can be usedto produce nearly an infinite array of conditioned media. In someembodiments of the present invention, a medium is conditioned byallowing differentiated pluripotent cells to be grown or maintained in amedium comprising about 1% to about 20% serum concentration. In otherembodiments, a medium is conditioned by allowing differentiatedpluripotent cells to be grown or maintained in a medium comprising about1 ng/ml to about 1000 ng/ml activin A. In still other embodiments, amedium is conditioned allowing differentiated pluripotent cells to begrown or maintained in a medium comprising about 1 ng/ml to about 1000ng/ml BMP4. In a preferred embodiment, a conditioned medium is preparedby allowing differentiated hESCs to be grown or maintained for 24 hoursin a medium, such as RPMI, comprising about 25 ng/ml activin A and about2 μM RA.

In some embodiments described herein, the cells used to condition themedium, which is used to enhance the differentiation of PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm, are cells thatare differentiated from pluripotent cells, such as hESCs, over about a 5day time period in a medium such as RPMI comprising about 0% to about20% serum and/or one or more growth/differentiation factors of the TGFβsuperfamily. Differentiation factors, such as activin A and BMP4 aresupplied at concentrations ranging from about 1 ng/ml to about 1000ng/ml. In certain embodiments of the present invention, the cells usedto condition the medium are differentiated from hESCs over about a 5 dayperiod in low serum RPMI. According to some embodiments, low serum RPMIrefers to a low serum containing medium, wherein the serum concentrationis gradually increased over a defined time period. For example, in oneembodiment, low serum RPMI comprises a concentration of about 0.2% fetalbovine serum (FBS) on the first day of cell growth, about 0).5% FBS onthe second day of cell growth and about 2% FBS on the third throughfifth day of cell growth. In another embodiment, low serum RPMIcomprises a concentration of about 0% on day one, about 0.2% on day twoand about 2% on days 3-6. In certain preferred embodiments, low serumRPMI is supplemented with one or more differentiation factors, such asactivin A and BMP4. In addition to its use in preparing cells used tocondition media, low serum RPMI can be used as a medium for thedifferentiation of PDX1-positive foregut endoderm cells fromPDX1-negative definitive endoderm cells.

It will be appreciated by those of ordinary skill in the art thatconditioned media can be prepared from media other than RPMI providedthat such media do not interfere with the growth or maintenance ofPDX1-positive foregut endoderm cells. It will also be appreciated thatthe cells used to condition the medium can be of various types. Inembodiments where freshly differentiated cells are used to condition amedium, such cells can be differentiated in a medium other than RPMIprovided that the medium does not inhibit the growth or maintenance ofsuch cells. Furthermore, a skilled artisan will appreciate that neitherthe duration of conditioning nor the duration of preparation of cellsused for conditioning is required to be 24 hours or 5 days,respectively, as other time periods will be sufficient to achieve theeffects reported herein.

In general, the use of a retinoid in combination with a fibroblastgrowth factor, a member of the TGFβ superfamily of growth factors, aconditioned medium or a combination of any of these foregutdifferentiation factors causes greater differentiation of PDX1-negativedefinitive endoderm to PDX1-positive foregut endoderm than the use of aretinoid alone. In a preferred embodiment, RA and FGF-10 are bothprovided to the PDX1-negative definitive endoderm cell culture. Inanother preferred embodiment, PDX1-negative definitive endoderm cellsare differentiated in a culture comprising a conditioned medium, activinA, activin B and RA.

With respect to some of the embodiments of differentiation processesdescribed herein, the above-mentioned foregut differentiation factorsare provided to the cells so that these factors are present in the cellculture or cell population at concentrations sufficient to promotedifferentiation of at least a portion of the PDX1-negative definitiveendoderm cell culture or cell population to PDX1-positive foregutendoderm cells. As defined previously, when used in connection with cellcultures and/or cell populations, the term “portion” means any non-zeroamount of the cell culture or cell population, which ranges from asingle cell to the entirety of the cell culture or cells population.

In some embodiments of processes described herein, a retinoid isprovided to the cells of a cell culture such that it is present at aconcentration of at least about 0.01 μM, at least about 0.02 μM, atleast about 0.04 μM, at least about 0.08 μM, at least about 0.1 μM, atleast about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, atleast about 0.5 μM, at least about 0.6 μM, at least about 0.7 μM, atleast about 0.8 μM, at least about 0.9 μM, at least about 1 μM, at leastabout 1.1 μM, at least about 1.2 μM, at least about 1.3 μM, at leastabout 1.4 μM, at least about 1.5 μM, at least about 1.6 μM, at leastabout 1.7 μM, at least about 1.8 μM, at least about 1.9 μM, at leastabout 2 μM, at least about 2.1 μM, at least about 2.2 μM, at least about2.3 μM, at least about 2.4 μM, at least about 2.5 μM, at least about 2.6μM, at least about 2.7 μM, at least about 2.8 μM, at least about 2.9 μM,at least about 3 μM, at least about 3.5 μM, at least about 4 μM, atleast about 4.5 μM, at least about 5 μM, at least about 10 μM, at leastabout 20 μM, at least about 30 μM, at least about 40 μM or at leastabout 50 μM. In a preferred embodiment, the retinoid is retinoic acid.

In other embodiments of the processes described herein, one or moredifferentiation factors of the fibroblast growth factor family arepresent in the cell culture. For example, in some embodiments, FGF-4 canbe present in the cell culture at a concentration of at least about 10ng/ml, at least about 25 ng/ml, at least about 50 ng/ml, at least about75 ng/ml, at least about 100 ng/ml, at least about 200 ng/ml, at leastabout 300 ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, orat least about 1000 ng/ml. In further embodiments, FGF-10 is present inthe cell culture at a concentration of at least about 10 ng/ml, at leastabout 25 ng/ml, at least about 50 ng/ml, at least about 75 ng/ml, atleast about 100 ng/ml, at least about 200 ng/ml, at least about 300ng/ml, at least about 400 ng/ml, at least about 500 ng/ml, or at leastabout 1000 ng/ml. In some embodiments, either FGF-4 or FGF-10, but notboth, is provided to the cell culture along with RA. In a preferredembodiment, RA is present in the cell culture at 1 μM and FGF-10 ispresent at a concentration of 50 ng/ml.

In some embodiments of the processes described herein, growth factors ofthe TGFβ superfamily and/or a conditioned medium are present in the cellculture. These differentiation factors can be used in combination withRA and/or other mid-foregut differentiation factors including, but notlimited to, FGF-4 and FGF-10. For example, in some embodiments, activinA and/or activin B can be present in the cell culture at a concentrationof at least about 5 ng/ml, at least about 10 ng/ml, at least about 25ng/ml, at least about 50 ng/ml, at least about 75 ng/ml, at least about100 ng/ml, at least about 200 ng/ml, at least about 300 ng/ml, at leastabout 400 ng/ml, at least about 500 ng/ml, or at least about 1000 ng/ml.In further embodiments, a conditioned medium is present in the cellculture at a concentration of at least about 1%, at least about 5%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 100% of the totalmedium. In some embodiments, activin A, activin B and a conditionedmedium are provided to the cell culture along with RA. In a preferredembodiment, PDX1-negative definitive endoderm cells are differentiatedto PDX1-positive foregut endoderm cells in cultures comprising about 1μM RA, about 25 ng/ml activin A and low serum RPMI medium that has beenconditioned for about 24 hours by differentiated hESCs, wherein thedifferentiated hESCs have been differentiated for about 5 days in lowserum RPMI comprising about 100 ng/ml activin A. In another preferredembodiment, activin B and/or FGF-10 are also present in the culture at25 ng/ml and 50 ng/ml, respectively.

In certain embodiments of the processes described herein, theabove-mentioned foregut differentiation factors are removed from thecell culture subsequent to their addition. For example, the foregutdifferentiation factors can be removed within about one day, about twodays, about three days, about four days, about five days, about sixdays, about seven days, about eight days, about nine days or about tendays after their addition.

Cultures of PDX1-positive foregut endoderm cells can be grown in amedium containing reduced serum. Serum concentrations can range fromabout 0.05% (v/v) to about 20% (v/v). In some embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement. For example, incertain embodiments, the serum concentration of the medium can be lessthan about 0.05% (v/v), less than about 0.1% (v/v), less than about 0.2%(v/v), less than about 0.3% (v/v), less than about 0.4% (v/v), less thanabout 0.5% (v/v), less than about 0.6% (v/v), less than about 0.7%(v/v), less than about 0.8% (v/v), less than about 0.9% (v/v), less thanabout 1% (v/v), less than about 2% (v/v), less than about 3% (v/v), lessthan about 4% (v/v), less than about 5% (v/v), less than about 6% (v/v),less than about 7% (v/v), less than about 8% (v/v), less than about 9%(v/v), less than about 10% (v/v), less than about 15% (v/v) or less thanabout 20% (v/v). In some embodiments, PDX1-positive foregut endodermcells are grown without serum. In other embodiments, PDX1-positiveforegut endoderm cells are grown with serum replacement.

In still other embodiments, PDX1-positive foregut endoderm cells aregrown in the presence of B27. In such embodiments, B27 can be providedto the culture medium in concentrations ranging from about 0.1% (v/v) toabout 20% (v/v) or in concentrations greater than about 20% (v/v). Incertain embodiments, the concentration of B27 in the medium is about0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3% (v/v), about 4%(v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v), about 8% (v/v),about 9% (v/v), about 10% (v/v), about 15% (v/v) or about 20% (v/v).Alternatively, the concentration of the added B27 supplement can bemeasured in terms of multiples of the strength of a commerciallyavailable B27 stock solution. For example, B27 is available fromInvitrogen (Carlsbad, Calif.) as a 50X stock solution. Addition of asufficient amount of this stock solution to a sufficient volume ofgrowth medium produces a medium supplemented with the desired amount ofB27. For example, the addition of 10 ml of 5× B27 stock solution to 90ml of growth medium would produce a growth medium supplemented with 5×B27. The concentration of B27 supplement in the medium can be about0.1×, about 0.2×, about 0.3×, about 0.4×, about 0.5×, about 0.6×, about0.7×, about 0.8×, about 0.9×, about 1×, about 1.1×, about 1.2×, about1.3×, about 1.4×, about 1.5×, about 1.6×, about 1.7×, about 1.8×, about1.9×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×,about 5×, about 6×, about 7×, about 8×, about 9×, about 10×, about 11 ×,about 12×, about 13×, about 14×, about 15×, about 16×, about 17×, about18×, about 19×, about 20× and greater than about 20×.

Monitoring the Differentiation of PDX1-Negative Definitive Endoderm toPDX1-Positive Endoderm

As with the differentiation of definitive endoderm cells frompluripotent cells, the progression of differentiation fromPDX1-negative, SOX17-positive definitive endoderm to PDX1-positiveforegut endoderm can be monitored by determining the expression ofmarkers characteristic of these cell types. Such monitoring permits oneto determine the amount of time that is sufficient for the production ofa desired amount of PDX1-positive foregut endoderm under variousconditions, for example, one or more differentiation factorconcentrations and environmental conditions. In preferred embodiments,the amount of time that is sufficient for the production of a desiredamount of PDX1-positive foregut endoderm is determined by detecting theexpression of PDX1. In some embodiments, the expression of certainmarkers is determined by detecting the presence or absence of themarker. Alternatively, the expression of certain markers can bedetermined by measuring the level at which the marker is present in thecells of the cell culture or cell population. In such embodiments, themeasurement of marker expression can be qualitative or quantitative. Asdescribed above, a preferred method of quantitating the expressionmarkers that are produced by marker genes is through the use of Q-PCR.In particular embodiments, Q-PCR is used to monitor the progression ofcells of the PDX1-negative, SOX17-positive definitive endoderm cultureto PDX1-positive foregut endoderm cells by quantitating expression ofmarker genes characteristic of PDX1-positive foregut endoderm and thelack of expression of marker genes characteristic of other cell types.Other methods which are known in the art can also be used to quantitatemarker gene expression. For example, the expression of a marker geneproduct can be detected by using antibodies specific for the marker geneproduct of interest. In some embodiments, the expression of marker genescharacteristic of PDX1-positive foregut endoderm as well as the lack ofsignificant expression of marker genes characteristic of PDX1-negativedefinitive endoderm, hESCs and other cell types is determined.

As described further in the Examples below, PDX1 is a marker gene thatis associated with PDX1-positive foregut endoderm. As such, in someembodiments of the processes described herein, the expression of PDX1 isdetermined. In other embodiments, the expression of other markers, whichare expressed in PDX1-positive foregut endoderm, including, but notlimited to, SOX17, HOXA13 and/or HOXC6 is also determined. Since PDX1can also be expressed by certain other cell types (that is, visceralendoderm and certain neural ectoderm), some embodiments described hereinrelate to demonstrating the absence or substantial absence of markergene expression that is associated with visceral endoderm and/or neuralectoderm. For example, in some embodiments, the expression of markers,which are expressed in visceral endoderm and/or neural cells, including,but not limited to, SOX7, AFP, SOX1, ZIC1 and/or NFM is determined.

In some embodiments, PDX1-positive foregut endoderm cell culturesproduced by the methods described herein are substantially free of cellsexpressing the SOX7, AFP, SOX1, ZIC1 or NFM marker genes. In certainembodiments, the PDX1-positive foregut endoderm cell cultures producedby the processes described herein are substantially free of visceralendoderm, parietal endoderm and/or neural cells.

Enrichment, Isolation and/or Purification of PDX1-Positive ForegutEndoderm

With respect to additional aspects of the processes described herein,PDX1-positive foregut endoderm cells can be enriched, isolated and/orpurified. In some embodiments, cell populations enriched forPDX1-positive foregut endoderm cells are produced by isolating suchcells from cell cultures.

In some embodiments of the processes described herein, PDX1-positiveforegut endoderm cells are fluorescently labeled then isolated fromnon-labeled cells by using a fluorescence activated cell sorter (FACS).In such embodiments, a nucleic acid encoding green fluorescent protein(GFP) or another nucleic acid encoding an expressible fluorescent markergene is used to label PDX1-positive cells. For example, in someembodiments, at least one copy of a nucleic acid encoding GFP or abiologically active fragment thereof is introduced into a pluripotentcell, preferably a human embryonic stem cell, downstream of the PDX1promoter such that the expression of the GFP gene product orbiologically active fragment thereof is under control of the PDX1promoter. In some embodiments, the entire coding region of the nucleicacid, which encodes PDX1, is replaced by a nucleic acid encoding GFP ora biologically active fragment thereof. In other embodiments, thenucleic acid encoding GFP or a biologically active fragment thereof isfused in frame with at least a portion of the nucleic acid encodingPDX1, thereby generating a fusion protein. In such embodiments, thefusion protein retains a fluorescent activity similar to GFP.

Fluorescently marked cells, such as the above-described pluripotentcells, are differentiated to definitive endoderm and then toPDX1-positive foregut endoderm as described previously above. BecausePDX1-positive foregut endoderm cells express the fluorescent markergene, whereas PDX1-negative cells do not, these two cell types can beseparated. In some embodiments, cell suspensions comprising a mixture offluorescently-labeled PDX1-positive cells and unlabeled PDX1-negativecells are sorted using a FACS. PDX1-positive cells are collectedseparately from PDX1-negative cells, thereby resulting in the isolationof such cell types. If desired, the isolated cell compositions can befurther purified by additional rounds of sorting using the same ordifferent markers that are specific for PDX1 -positve foregut endoderm.

In addition to the procedures just described, PDX1-positive foregutendoderm cells may also be isolated by other techniques for cellisolation. Additionally, PDX1-positive foregut endoderm cells may alsobe enriched or isolated by methods of serial subculture in growthconditions which promote the selective survival or selective expansionof said PDX1-positive foregut endoderm cells.

It will be appreciated that the above-described enrichment, isolationand purification procedures can be used with such cultures at any stageof differentiation.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of PDX1-positive foregut endoderm cells and/or tissues canbe produced ill vitro from PDX1-negative, SOX17-positive definitiveendoderm cell cultures or cell populations which have undergone at leastsome differentiation. In some embodiments, the cells undergo randomdifferentiation. In a preferred embodiment, however, the cells aredirected to differentiate primarily into PDX1-positive foregut endodermcells. Some preferred enrichment, isolation and/or purification methodsrelate to the in vitro production of PDX1-positive foregut endodermcells from human embryonic stem cells.

Using the methods described herein, cell populations or cell culturescan be enriched in PDX1-positive foregut endoderm cell content by atleast about 2- to about 1000-fold as compared to untreated cellpopulations or cell cultures. In some embodiments, PDX1-positive foregutendoderm cells can be enriched by at least about 5- to about 500-fold ascompared to untreated cell populations or cell cultures. In otherembodiments, PDX1-positive foregut endoderm cells can be enriched fromat least about 10- to about 200-fold as compared to untreated cellpopulations or cell cultures. In still other embodiments, PDX1-positiveforegut endoderm cells can be enriched from at least about 20- to about100-fold as compared to untreated cell populations or cell cultures. Inyet other embodiments, PDX1-positive foregut endoderm cells can beenriched from at least about 40- to about 80-fold as compared tountreated cell populations or cell cultures. In certain embodiments,PDX1-positive foregut endoderm cells can be enriched from at least about2- to about 20-fold as compared to untreated cell populations or cellcultures.

Compositions Comprising PDX1-Positive Foregut Endoderm

Some embodiments described herein relate to cell compositions, such ascell cultures or cell populations, comprising PDX1-positive endodermcells, wherein the PDX1-positive endoderm cells are multipotent cellsthat can differentiate into cells, tissues or organs derived from theanterior portion of the gut tube (PDX1-positive foregut endoderm). Inaccordance with certain embodiments, the PDX1-positive foregut endodermare mammalian cells, and in a preferred embodiment, these cells arehuman cells.

Other embodiments described herein relate to compositions, such as cellcultures or cell populations, comprising cells of one or more cell typesselected from the group consisting of hESCs, PDX1-negative definitiveendoderm cells, PDX1-positive foregut endoderm cells and mesoderm cells.In some embodiments, hESCs comprise less than about 5%, less than about4%, less than about 3%, less than about 2% or less than about 1% of thetotal cells in the culture. In other embodiments, PDX1-negativedefinitive endoderm cells comprise less than about 90%, less than about85%, less than about 80%, less than about 75%, less than about 70%, lessthan about 65%, less than about 60%, less than about 55%, less thanabout 50%, less than about 45%, less than about 40%, less than about35%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 12%, less than about 10%, less thanabout 8%, less than about 6%, less than about 5%, less than about 4%,less than about 3%, less than about 2% or less than about 1% of thetotal cells in the culture. In yet other embodiments, mesoderm cellscomprise less than about 90%, less than about 85%, less than about 80%,less than about 75%, less than about 70%, less than about 65%, less thanabout 60%, less than about 55%, less than about 50%, less than about45%, less than about 40%, less than about 35%, less than about 30%, lessthan about 25%, less than about 20%, less than about 15%, less thanabout 12%, less than about 10%, less than about 8%, less than about 6%,less than about 5%, less than about 4%, less than about 3%, less thanabout 2% or less than about 1% of the total cells in the culture.

Additional embodiments described herein relate to compositions, such ascell cultures or cell populations, produced by the processes describedherein, which comprise PDX1-positive foregut endoderm as the majoritycell type. In some embodiments, the processes described herein producecell cultures and/or cell populations comprising at least about 99%, atleast about 98%, at least about 97%, at least about 96%, at least about95%, at least about 94%, at least about 93%, at least about 92%, atleast about 91%, at least about 90%, at least about 85%, at least about80%, at least about 75%, at least about 70%, at least about 65%, atleast about 60%, at least about 55%, at least about 54%, at least about53%, at least about 52% or at least about 51% PDX1-positive foregutendoderm cells. In preferred embodiments the cells of the cell culturesor cell populations comprise human cells. In other embodiments, theprocesses described herein produce cell cultures or cell populationscomprising at least about 50%, at least about 45%, at least about 40%,at least about 35%, at least about 30%, at least about 25%, at leastabout 24%, at least about 23%, at least about 22%, at least about 21%,at least about 20%, at least about 19%, at least about 18%, at leastabout 17%, at least about 16%, at least about 15%, at least about 14%,at least about 13%, at least about 12%, at least about 11%, at leastabout 10%, at least about 9%, at least about 8%, at least about 7%, atleast about 6%, at least about 5%, at least about 4%, at least about 3%,at least about 2% or at least about 1% PDX1-positive foregut endodermcells. In preferred embodiments, the cells of the cell cultures or cellpopulations comprise human cells. In some embodiments, the percentage ofPDX1-positive foregut endoderm cells in the cell cultures or populationsis calculated without regard to the feeder cells remaining in theculture.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mixtures of PDX1-positiveforegut endoderm cells and PDX1-negative definitive endoderm cells. Forexample, cell cultures or cell populations comprising at least about 5PDX1 -positive foregut endoderm cells for about every 95 PDX1-negativedefinitive endoderm cells can be produced. In other embodiments, cellcultures or cell populations comprising at least about 95 PDX1-positiveforegut endoderm cells for about every 5 PDX1-negative definitiveendoderm cells can be produced. Additionally, cell cultures or cellpopulations comprising other ratios of PDX1-positive foregut endodermcells to PDX1-negative definitive endoderm cells are contemplated. Forexample, compositions comprising at least about 1 PDX1-positive foregutendoderm cell for about every 1,000,000 PDX1-negative definitiveendoderm cells, at least about 1 PDX1-positive foregut endoderm cell forabout every 100,000 PDX1-negative definitive endoderm cells, at leastabout 1 PDX1-positive foregut endoderm cell for about every 10,000PDX1-negative definitive endoderm cells, at least about 1 PDX1-positiveforegut endoderm cell for about every 1000 PDX1-negative definitiveendoderm cells, at least about 1 PDX1-positive foregut endoderm cell forabout every 500 PDX1-negative definitive endoderm cells, at least about1 PDX1-positive foregut endoderm cell for about every 100 PDX1-negativedefinitive endoderm cells, at least about 1 PDX1-positive foregutendoderm cell for about every 10 PDX1-negative definitive endodermcells, at least about 1 PDX1-positive foregut endoderm cell for aboutevery 5 PDX1-negative definitive endoderm cells, at least about 1PDX1-positive foregut endoderm cell for about every 4 PDX1 -negativedefinitive endoderm cells, at least about 1PDX1-positive foregutendoderm cell for about every 2 PDX1-negative definitive endoderm cells,at least about 1 PDX-1 positive foregut endoderm cell for about every 1PDX1-negative definitive endoderm cell, at least about 2 PDX1-positiveforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell, at least about 4 PDX1-positive foregut endoderm cells forabout every 1 PDX1-negative definitive endoderm cell, at least about 5PDX1-positive foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 10 PDX1-positive foregutendoderm cells for about every 1 PDX1-negative definitive endoderm cell,at least about 20 PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 50 PDX1-positiveforegut endoderm cells for about every 1 PDX1-negative definitiveendoderm cell, at least about 100 PDX1-positive foregut endoderm cellsfor about every 1 PDX1-negative definitive endoderm cell, at least about1000 PDX1-positive foregut endoderm cells for about every 1PDX1-negative definitive endoderm cell, at least about 10,000PDX1-positive foregut endoderm cells for about every 1 PDX1-negativedefinitive endoderm cell, at least about 100,000 PDX1-positive foregutendoderm cells for about every 1 PDX1 -negative definitive endoderm celland at least about 1,000,000 PDX1-positive foregut endoderm cells forabout every 1 PDX1-negative definitive endoderm cell are contemplated.

In some embodiments described herein, the PDX1-negative definitiveendoderm cells from which PDX1-positive foregut endoderm cells areproduced are derived from human pluripotent cells, such as humanpluripotent stem cells. In certain embodiments, the human pluripotentcells are derived from a morula, the inner cell mass of an embryo or thegonadal ridges of an embryo. In certain other embodiments, the humanpluripotent cells are derived from the gonadal or germ tissues of amulticellular structure that has developed past the embryonic stage.

Further embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising human cells, includinghuman PDX1-positive foregut endoderm, wherein the expression of the PDX1marker is greater than the expression of the AFP, SOX7, SOX1, ZIC1and/or NFM marker in at least about 2% of the human cells. In otherembodiments, the expression of the PDX1 marker is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the human cells, in at least about 10% of the human cells,in at least about 15% of the human cells, in at least about 20% of thehuman cells, in at least about 25% of the human cells, in at least about30% of the human cells, in at least about 35% of the human cells, in atleast about 40% of the human cells, in at least about 45% of the humancells, in at least about 50% of the human cells, in at least about 55%of the human cells, in at least about 60% of the human cells, in atleast about 65% of the human cells, in at least about 70% of the humancells, in at least about 75% of the human cells, in at least about 80%of the human cells, in at least about 85% of the human cells, in atleast about 90% of the human cells, in at least about 95% of the humancells or in at least about 98% of the human cells. In some embodiments,the percentage of human cells in the cell cultures or populations,wherein the expression of PDX1 is greater than the expression of theAFP, SOX7, SOX1, ZIC1 and/or NFM marker, is calculated without regard tofeeder cells.

It will be appreciated that some embodiments described herein relate tocompositions, such as cell cultures or cell populations, comprisinghuman PDX1-positive foregut endoderm cells, wherein the expression ofone or more markers selected from the group consisting of SOX17, HOXA 13and HOXC6 is greater than the expression of the AFP, SOX7, SOX1, ZIC1and/or NFM marker in from at least about 2% to greater than at leastabout 98% of the human cells. In some embodiments, the expression of oneor more markers selected from the group consisting of SOX17, HOXA13 andHOXC6 is greater than the expression of the AFP, SOX7, SOX1, ZIC 1and/or NFM marker in at least about 5% of the human cells, in at leastabout 10% of the human cells, in at least about 15% of the human cells,in at least about 20% of the human cells, in at least about 25% of thehuman cells, in at least about 30% of the human cells, in at least about35% of the human cells, in at least about 40% of the human cells, in atleast about 45% of the human cells, in at least about 50% of the humancells, in at least about 55% of the human cells, in at least about 60%of the human cells, in at least about 65% of the human cells, in atleast about 70% of the human cells, in at least about 75% of the humancells, in at least about 80% of the human cells, in at least about 85%of the human cells, in at least about 90% of the human cells, in atleast about 95% of the human cells or in at least about 98% of the humancells. In some embodiments, the percentage of human cells in the cellcultures or populations, wherein the expression of one or more markersselected from the group consisting of SOX17, HOXA13 and HOXC6 is greaterthan the expression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker, iscalculated without regard to feeder cells.

Additional embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endoderm cells, wherein the expression of the PDX1marker is greater than the expression of the AFP, SOX7, SOX1,ZIC1 and/orNFM marker in at least about 2% of the endodermal cells. In otherembodiments, the expression of the PDX1 marker is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the endodermal cells, in at least about 10% of theendodermal cells, in at least about 15% of the endodermal cells, in atleast about 20% of the endodermal cells, in at least about 25% of theendodermal cells, in at least about 30% of the endodermal cells, in atleast about 35% of the endodermal cells, in at least about 40% of theendodermal cells, in at least about 45% of the endodermal cells, in atleast about 50% of the endodermal cells, in at least about 55% of theendodermal cells, in at least about 60% of the endodermal cells, in atleast about 65% of the endodermal cells, in at least about 70% of theendodermal cells, in at least about 75% of the endodermal cells, in atleast about 80% of the endodermal cells, in at least about 85% of theendodermal cells, in at least about 90% of the endodermal cells, in atleast about 95% of the endodermal cells or in at least about 98% of theendodermal cells.

Still other embodiments described herein relate to compositions, such ascell cultures or cell populations, comprising mammalian endodermalcells, such as human endodermal cells, wherein the expression of one ormore markers selected from the group consisting of SOX17, HOXA13 andHOXC6 is greater than the expression of the AFP, SOX7, SOX1, ZIC 1and/or NFM marker in at least about 2% of the endodermal cells. In otherembodiments, the expression of one or more markers selected from thegroup consisting of SOX17, HOXA13 and HOXC6 is greater than theexpression of the AFP, SOX7, SOX1, ZIC1 and/or NFM marker in at leastabout 5% of the endodermal cells, in at least about 10% of theendodermal cells, in at least about 15% of the endodermal cells, in atleast about 20% of the endodermal cells, in at least about 25% of theendodermal cells, in at least about 30% of the endodermal cells, in atleast about 35% of the endodermal cells, in at least about 40% of theendodermal cells, in at least about 45% of the endodermal cells, in atleast about 50% of the endodermal cells, in at least about 55% of theendodermal cells, in at least about 60% of the endodermal cells, in atleast about 65% of the endodermal cells, in at least about 70% of theendodermal cells, in at least about 75% of the endodermal cells, in atleast about 80% of the endodermal cells, in at least about 85% of theendodermal cells, in at least about 90% of the endodermal cells, in atleast about 95% of the endodermal cells or at least about 98% of theendodermal cells.

Using the processes described herein, compositions comprisingPDX1-positive foregut endoderm cells substantially free of other celltypes can be produced. In some embodiments of the present invention, thePDX1-positive foregut endoderm cell populations or cell culturesproduced by the methods described herein are substantially free of cellsthat significantly express the AFP, SOX7, SOX1, ZIC1 and/or NFM markergenes.

In one embodiment, a description of a PDX1-positive foregut endodermcell based on the expression of marker genes is, PDX1 high, AFP low,SOX7 low, SOX1 low, ZIC1 low and NFM low.

Increasing Expression of PDX1 in a SOX17-Positive Definitive EndodermCell

Some aspects of the processes described herein are related to methods ofincreasing the expression of the PDX1 gene product in cell cultures orcell populations comprising SOX17-positive definitive endoderm cells. Insuch embodiments, the SOX17-positive definitive endoderm cells arecontacted with a differentiation factor in an amount that is sufficientto increase the expression of the PDX1 gene product. The SOX17-positivedefinitive endoderm cells that are contacted with the differentiationfactor can be either PDX1-negative or PDX1-positive. In someembodiments, the differentiation factor can be a retinoid. In certainembodiments, SOX17-positive definitive endoderm cells are contacted witha retinoid at a concentration ranging from about 0.01 μM to about 50 μM.In a preferred embodiment, the retinoid is RA.

In other embodiments of the processes described herein, the expressionof the PDX1 gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with a differentiation factor of the fibroblastgrowth factor family. Such differentiation factors can either be usedalone or in conjunction with RA. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with a fibroblast growth factorat a concentration ranging from about 10 ng/ml to about 1000 ng/ml. In apreferred embodiment, the FGF growth factor is FGF-10.

In some embodiments of the processes described herein, the expression ofthe PDX1 gene product in cell cultures or cell populations comprisingSOX17-positive definitive endoderm cells is increased by contacting theSOX17-positive cells with B27. This differentiation factor can either beused alone or in conjunction with one or both of retinoid and FGF familydifferentiation factors. In some embodiments, the SOX17-positivedefinitive endoderm cells are contacted with B27 at a concentrationranging from about 0.1% (v/v) to about 20% (v/v). In a preferredembodiment, the SOX17-positive definitive endoderm cells are contactedwith RA, FGF-10 and B27.

Methods for increasing the expression of the PDX1 gene product in cellcultures or cell populations comprising SOX17-positive definitiveendoderm cells can be carried out in growth medium containing reduced orno serum. In some embodiments, serum concentrations range from about0.05% (v/v) to about 20% (v/v). In some embodiments, the SOX17-positivecells are grown with serum replacement.

Identification of Factors Capable of Promoting the Differentiation ofDefinitive Endoderm Cells

Certain screening methods described herein relate to methods foridentifying at least one differentiation factor that is capable ofpromoting the differentiation of definitive endoderm cells. In someembodiments of these methods, cell populations comprising definitiveendoderm cells, such as human definitive endoderm cells, are obtained.The cell population is then provided with a candidate differentiationfactor. At a first time point, which is prior to or at approximately thesame time as providing the candidate differentiation factor, expressionof a marker is determined. Alternatively, expression of the marker canbe determined after providing the candidate differentiation factor. At asecond time point, which is subsequent to the first time point andsubsequent to the step of providing the candidate differentiation factorto the cell population, expression of the same marker is againdetermined. Whether the candidate differentiation factor is capable ofpromoting the differentiation of the definitive endoderm cells isdetermined by comparing expression of the marker at the first time pointwith the expression of the marker at the second time point. Ifexpression of the marker at the second time point is increased ordecreased as compared to expression of the marker at the first timepoint, then the candidate differentiation factor is capable of promotingthe differentiation of definitive endoderm cells.

Some embodiments of the screening methods described herein utilize cellpopulations or cell cultures which comprise human definitive endodermcells. For example, the cell population can be a substantially purifiedpopulation of human definitive endoderm cells. Alternatively, the cellpopulation can be an enriched population of human definitive endodermcells, wherein at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97% or greater than at least about 97%of the human cells in the cell population are human definitive endodermcells. In other embodiments described herein, the cell populationcomprises human cells wherein at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85% orgreater than at least about 85% of the human cells are human definitiveendoderm cells. In some embodiments, the cell population includesnon-human cells such as non-human feeder cells. In other embodiments,the cell population includes human feeder cells. In such embodiments, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95% or greater than at least about 95% of the human cells, other thansaid feeder cells, are human definitive endoderm cells. In someembodiments of the screening methods described herein, the cellpopulations further comprise PDX1-positive endoderm cells including, butnot limited to, PDX1-positive foregut endoderm cells.

In embodiments of the screening methods described herein, the cellpopulation is contacted or otherwise provided with a candidate (test)differentiation factor. The candidate differentiation factor cancomprise any molecule that may have the potential to promote thedifferentiation of human definitive endoderm cells. In some embodimentsdescribed herein, the candidate differentiation factor comprises amolecule that is known to be a differentiation factor for one or moretypes of cells. In alternate embodiments, the candidate differentiationfactor comprises a molecule that in not known to promote celldifferentiation. In preferred embodiments, the candidate differentiationfactor comprises molecule that is not known to promote thedifferentiation of human definitive endoderm cells.

In some embodiments of the screening methods described herein, thecandidate differentiation factor comprises a small molecule. Inpreferred embodiments, a small molecule is a molecule having a molecularmass of about 10,000 amu or less. In some embodiments, the smallmolecule comprises a retinoid. In some embodiments, the small moleculecomprises retinoic acid.

In other embodiments described herein, the candidate differentiationfactor comprises a polypeptide. The polypeptide can be any polypeptideincluding, but not limited to, a glycoprotein, a lipoprotein, anextracellular matrix protein, a cytokine, a chemokine, a peptidehormone, an interleukin or a growth factor. Preferred polypeptidesinclude growth factors. In some preferred embodiments, the candidatedifferentiation factors comprises one or more growth factors selectedfrom the group consisting of FGF10, FGF4, FGF2 and Wnt3B.

In some embodiments of the screening methods described herein, thecandidate differentiation factors comprise one or more growth factorsselected from the group consisting of Amphiregulin, B-lymphocytestimulator, IL-16, Thymopoietin, TRAIL/Apo-2, Pre B cell colonyenhancing factor, Endothelial differentiation-related factor 1 (EDF1),Endothelial monocyte activating polypeptide II, Macrophage migrationinhibitory factor (MIF), Natural killer cell enhancing factor (NKEFA),Bone mophogenetic protein 2, Bone mophogenetic protein 8 (osteogeneicprotein 2), Bone morphogenic protein 6, Bone morphogenic protein 7,Connective tissue growth factor (CTGF), CGI-149 protein (neuroendocrinedifferentiation factor), Cytokine A3 (macrophage inflammatory protein1-alpha), Gliablastoma cell differentiation-related protein (GBDR1),Hepatoma-derived growth factor, Neuromedin U-25 precursor, Vascularendothelial growth factor (VEGF), Vascular endothelial growth factor B(VEGF-B), T-cell specific RANTES precursor, thymic dendriticcell-derived factor 1, Transferrin, Interleukin-1 (IL 1), Interleukin-2(IL 2), Interleukin-3 (IL 3), Interleukin-4 (IL 4), Interleukin-5 (IL5), Interleukin-6 (IL 6), Interleukin-7 (IL 7), Interleukin-8 (IL 8),Interleukin-9 (IL 9), Interleukin-10 (IL 10), Interleukin-11 (IL 11),Interleukin-12 (IL 12), Interleukin-13 (IL 13), Granulocyte-colonystimulating factor (G-CSF), Granulocyte macrophage colony stimulatingfactor (GM-CSF), Macrophage colony stimulating factor (M-CSF),Erythropoietin, Thrombopoietin, Vitamin D₃, Epidermal growth factor(EGF), Brain-derived neurotrophic factor, Leukemia inhibitory factor,Thyroid hormone, Basic fibroblast growth factor (bFGF), aFGF, FGF-4,FGF-6, Keratinocyte growth factor (KGF), Platelet-derived growth factor(PDGF), Platelet-derived growth factor-BB, beta nerve growth factor,activin A, Transforming growth factor beta 1 (TGF-β1), Interferon-a,Interferon-β, Interferon-?, Tumor necrosis factor-a, Tumor necrosisfactor-β, Burst promoting activity (BPA), Erythroid promoting activity(EPA), PGE₂, insulin growth factor-1 (IGF-1), IGF-II, Neutrophin growthfactor (NGF), Neutrophin-3, Neutrophin 4/5, Ciliary neurotrophic factor,Glial-derived nexin, Dexamethasone, β-mercaptoethanol, Retinoic acid,Butylated hydroxyanisole, 5-azacytidine, Amphotericin B, Ascorbic acid,Ascrorbate, isobutylxanthine, indomethacin, β-glycerolphosphate,nicotinamide, DMSO, Thiazolidinediones, TWS119, oxytocin, vasopressin,melanocyte-stimulating hormone, corticortropin, lipotropin, thyrotropin,growth hormone, prolactin, luteinizing hormone, human chorionicgonadotropin, follicle stimulating hormone, corticotropin-releasingfactor, gonadotropin-releasing factor, prolactin-releasing factor,prolactin-inhibiting factor, growth-hormone releasing factor,somatostatin, thyrotropin-releasing factor, calcitonin gene-relatedpeptide, parathyroid hormone, glucagon-like peptide 1, glucose-dependentinsulinotropic polypeptide, gastrin, secretin, cholecystokinin, motilin,vasoactive intestinal peptide, substance P, pancreatic polypeptide,peptide tyrosine tyrosine, neuropeptide tyrosine, insulin, glucagon,placental lactogen, relaxin, angiotensin II, calctriol, atrialnatriuretic peptide, and melatonin. thyroxine, triiodothyronine,calcitonin, estradiol, estrone, progesterone, testosterone, cortisol,corticosterone, aldosterone, epinephrine, norepinepherine, androstiene,calcitriol, collagen, Dexamethasone, β-mercaptoethanol, Retinoic acid,Butylated hydroxyanisole, 5-azacytidine, Amphotericin B, Ascorbic acid,Ascrorbate, isobutylxanthine, indomethacin, β-glycerolphosphate,nicotinamide, DMSO, Thiazolidinediones, and TWS119.

In some embodiments of the screening methods described herein, thecandidate differentiation factor is provided to the cell population inone or more concentrations. In some embodiments, the candidatedifferentiation factor is provided to the cell population so that theconcentration of the candidate differentiation factor in the mediumsurrounding the cells ranges from about 0.1 ng/ml to about 10 mg/ml. Insome embodiments, the concentration of the candidate differentiationfactor in the medium surrounding the cells ranges from about 1 ng/ml toabout 1 mg/ml. In other embodiments, the concentration of the candidatedifferentiation factor in the medium surrounding the cells ranges fromabout 10 ng/ml to about 100 μg/ml. In still other embodiments, theconcentration of the candidate differentiation factor in the mediumsurrounding the cells ranges from about 100 ng/ml to about 10 μg/ml. Inpreferred embodiments, the concentration of the candidatedifferentiation factor in the medium surrounding the cells is about 5ng/ml, about 25 ng/ml, about 50 ng/ml, about 75 ng/ml, about 100 ng/ml,about 125 ng/ml, about 150 ng/ml, about 175 ng/ml, about 200 ng/ml,about 225 ng/ml, about 250 ng/ml, about 275 ng/ml, about 300 ng/ml,about 325 ng/ml, about 350 ng/ml, about 375 ng/ml, about 400 ng/ml,about 425 ng/ml, about 450 ng/ml, about 475 ng/ml, about 500 ng/ml,about 525 ng/ml, about 550 ng/ml, about 575 ng/ml, about 600 ng/ml,about 625 ng/ml, about 650 ng/ml, about 675 ng/ml, about 700 ng/ml,about 725 ng/ml, about 750 ng/ml, about 775 ng/ml, about 800 ng/ml,about 825 ng/ml, about 850 ng/ml, about 875 ng/ml, about 900 ng/ml,about 925 ng/ml, about 950 ng/ml, about 975 ng/ml, about 1 μg/ml, about2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 6 μg/ml,about 7 μg/ml, about 8 μg/ml, about 9 μg/ml, about 10 μg/ml, about 11μg/ml, about 12 μg/ml, about 13 μg/ml, about 14 μg/ml, about 15 μg/ml,about 16 μg/ml, about 17 μg/ml, about 18 μg/ml, about 19 μg/ml, about 20μg/ml, about 25 μg/ml, about 50 μg/ml, about 75 μg/ml, about 100 μg/ml,about 125 μg/ml, about 150 μg/ml, about 175 μg/ml, about 200 μg/ml,about 250 μg/ml, about 300 μg/ml, about 350 μg/ml, about 400 μg/ml,about 450 μg/ml, about 500 μg/ml, about 550 μg/ml, about 600 μg/ml,about 650 μg/ml, about 700 μg/ml, about 750 μg/ml, about 800 μg/ml,about 850 μg/ml, about 900 μg/ml, about 950 μg/ml, about 1000 μg/ml orgreater than about 1000 μg/ml.

In certain embodiments of the screening methods described herein, thecell population is provided with a candidate differentiation factorwhich comprises any molecule other than foregut differentiation factor.For example, in some embodiments, the cell population is provided with acandidate differentiation factor which comprises any molecule other thana retinoid, a member of the TGFβ superfamily of growth factors, FGF10 orFGF4. In some embodiments, the cell population is provided with acandidate differentiation factor which comprises any molecule other thanretinoic acid.

In some embodiments, steps of the screening methods described hereincomprise determining expression of at least one marker at a first timepoint and a second time point. In some of these embodiments, the firsttime point can be prior to or at approximately the same time asproviding the cell population with the candidate differentiation factor.Alternatively, in some embodiments, the first time point is subsequentto providing the cell population with the candidate differentiationfactor. In some embodiments, expression of a plurality of markers isdetermined at a first time point.

In addition to determining expression of at least one marker at a firsttime point, some embodiments of the screening methods described hereincontemplate determining expression of at least one marker at a secondtime point, which is subsequent to the first time point and which issubsequent to providing the cell population with the candidatedifferentiation factor. In such embodiments, expression of the samemarker is determined at both the first and second time points. In someembodiments, expression of a plurality of markers is determined at boththe first and second time points. In such embodiments, expression of thesame plurality of markers is determined at both the first and secondtime points. In some embodiments, marker expression is determined at aplurality of time points, each of which is subsequent to the first timepoint, and each of which is subsequent to providing the cell populationwith the candidate differentiation factor. In certain embodiments,marker expression is determined by Q-PCR. In other embodiments, markerexpression is determined by immunocytochemistry.

In certain embodiments of the screening methods described herein, themarker having its expression is determined at the first and second timepoints is a marker that is associated with the differentiation of humandefinitive endoderm cells to cells which are the precursors of cellswhich make up tissues and/or organs that are derived from the gut tube.In some embodiments, the tissues and/or organs that are derived from thegut tube comprise terminally differentiated cells. In some embodiments,the marker is indicative of pancreatic cells or pancreatic precursorcells. In preferred embodiments, the marker is pancreatic-duodenalhomeobox factor-1 (PDX1). In other embodiments, the marker is homeoboxA13 (HOXA13) or homeobox C6 (HOXC6). Additionally, in other embodiments,the marker is indicative of liver cells or liver precursor cells. Incertain preferred embodiments, the marker is albumin, hepatocytespecific antigen (HSA) or prospero-related homeobox 1 (PROX1). In otherembodiments, the marker is indicative of lung or lung precursor cells.In some preferred embodiments, the marker is thyroid transcriptionfactor 1 (TITF1). In yet other embodiments, the marker is indicative ofintestinal or intestinal precursor cells. In additional preferredembodiments, the marker is villin, glucose transporter-2 (GLUT2),apolipoprotein A1 (APOA1), vascular cell adhesion molecule-1 (VACM1),von Willebrand factor (VWF), CXC-type chemokine receptor 4 (CXCR4) orcaudal type homeobox transcription factor 2 (CDX2). In still otherembodiments, the marker is indicative of stomach or stomach precursorcells. In additional preferred embodiments, the marker is VCAM1, VWF orCXCR4. In other embodiments, the marker is indicative of thyroid orthyroid precursor cells. In such embodiments, the marker is TITF1. Instill other embodiments, the marker is indicative of thymus or thymusprecursor cells.

In some embodiments of the screening methods described herein,sufficient time is allowed to pass between providing the cell populationwith the candidate differentiation factor and determining markerexpression at the second time point. Sufficient time between providingthe cell population with the candidate differentiation factor anddetermining expression of the marker at the second time point can be aslittle as from about 1 hour to as much as about 10 days. In someembodiments, the expression of at least one marker is determinedmultiple times subsequent to providing the cell population with thecandidate differentiation factor. In some embodiments, sufficient timeis at least about 1 hour, at least about 6 hours, at least about 12hours, at least about 18 hours, at least about 24 hours, at least about30 hours, at least about 36 hours, at least about 42 hours, at leastabout 48 hours, at least about 54 hours, at least about 60 hours, atleast about 66 hours, at least about 72 hours, at least about 78 hours,at least about 84 hours, at least about 90 hours, at least about 96hours, at least about 102 hours, at least about 108 hours, at leastabout 114 hours, at least about 120 hours, at least about 126 hours, atleast about 132 hours, at least about 138 hours, at least about 144hours, at least about 150 hours, at least about 156 hours, at leastabout 162 hours, at least about 168 hours, at least about 174 hours, atleast about 180 hours, at least about 186 hours, at least about 192hours, at least about 198 hours, at least about 204 hours, at leastabout 210 hours, at least about 216 hours, at least about 222 hours, atleast about 228 hours, at least about 234 hours or at least about 240hours.

In some embodiments of the methods described herein, it is furtherdetermined whether the expression of the marker at the second time pointhas increased or decreased as compared to the expression of this markerat the first time point. An increase or decrease in the expression ofthe at least one marker indicates that the candidate differentiationfactor is capable of promoting the differentiation of the definitiveendoderm cells. Similarly, if expression of a plurality of markers isdetermined, it is further determined whether the expression of theplurality of markers at the second time point has increased or decreasedas compared to the expression of this plurality of markers at the firsttime point. An increase or decrease in marker expression can bedetermined by measuring or otherwise evaluating the amount, level oractivity of the marker in the cell population at the first and secondtime points. Such determination can be relative to other markers, forexample housekeeping gene expression, or absolute. In certainembodiments, wherein marker expression is increased at the second timepoint as compared with the first time point, the amount of increase isat least about 2-fold, at least about 5-fold, at least about 10-fold, atleast about 20-fold, at least about 30-fold, at least about 40-fold, atleast about 50-fold, at least about 60-fold, at least about 70-fold, atleast about 80-fold, at least about 90-fold, at least about 100-fold ormore than at least about 100-fold. In some embodiments, the amount ofincrease is less than 2-fold. In embodiments where marker expression isdecreased at the second time point as compared with the first timepoint, the amount of decrease is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 20-fold, at least about30-fold, at least about 40-fold, at least about 50-fold, at least about60-fold, at least about 70-fold, at least about 80-fold, at least about90-fold, at least about 100-fold or more than at least about 100-fold.In some embodiments, the amount of decrease is less than 2-fold.

In some embodiments of the screening methods described herein, afterproviding the cell population with a candidate differentiation factor,the human definitive endoderm cells differentiate into one or more celltypes of the definitive endoderm lineage. In some embodiments, afterproviding the cell population with a candidate differentiation factor,the human definitive endoderm cells differentiate into cells that arederived from the gut tube. Such cells include, but are not limited to,cells of the pancreas, liver, lungs, stomach, intestine, thyroid,thymus, pharynx, gallbladder and urinary bladder as well as precursorsof such cells. Additionally, these cells can further develop into higherorder structures such as tissues and/or organs.

It will be appreciated that screening methods similar to those describedabove can be used to identify one or more differentiation factorscapable of promoting the differentiation of human PDX1-positive endodermcells in a cell population which comprises human PDX1-positive endodermcells. In certain embodiments, the human PDX1-positive endoderm cellsare PDX1-positive foregut/midgut endoderm cells. In preferredembodiments, the human PDX1-positive endoderm cells are PDX1-positiveforegut endoderm cells. In other preferred embodiments, the humanPDX1-positive endoderm cells are PDX1-positive endoderm cells of theposterior portion of the foregut. In especially preferred embodiments,the human PDX1-positive foregut endoderm cells are multipotent cellsthat can differentiate into cells, tissues or organs derived from theanterior portion of the gut tube.

Identification of Factors Capable of Promoting the Differentiation ofPDX1-Negative Definitive Endoderm Cells to PDX1-Positive ForegutEndoderm Cells

Aspects of the screening methods described herein relate to methods ofidentifying one or more differentiation factors capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In such methods, a cell culture orcell population comprising PDX1-negative definitive endoderm cells isobtained and the expression of PDX1 in the cell culture or cellpopulation is determined. After determining the expression of PDX1, thecells of the cell culture or cell population are contacted with acandidate differentiation factor. In some embodiments, the expression ofPDX1 is determined at the time of contacting or shortly after contactingthe cells with a candidate differentiation factor. PDX1 expression isthen determined at one or more times after contacting the cells with thecandidate differentiation factor. If the expression of PDX1 hasincreased after contact with the candidate differentiation factor ascompared to PDX1 expression prior to contact with the candidatedifferentiation factor, the candidate differentiation factor isidentified as capable of promoting the differentiation of PDX1-negativedefinitive endoderm cells to PDX1-positive foregut endoderm cells.

In some embodiments, the above-described methods of identifying factorscapable of promoting the differentiation of PDX1-negative definitiveendoderm cells to PDX1-positive foregut endoderm cells also includedetermining the expression of the HOXA13 gene and/or the HOXC6 gene inthe cell culture or cell population. In such embodiments, the expressionof HOXA13 and/or HOXC6 is determined both before and after the cells arecontacted with the candidate differentiation factor. If the expressionof PDX1 and HOXA13 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXA13 expression priorto contact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. Similarly, if the expression ofPDX1 and HOXC6 has increased after contact with the candidatedifferentiation factor as compared to PDX1 and HOXC6 expression prior tocontact with the candidate differentiation factor, the candidatedifferentiation factor is identified as capable of promoting thedifferentiation of PDX1-negative definitive endoderm cells toPDX1-positive foregut endoderm cells. In a preferred embodiment, acandidate differentiation factor is identified as being capable ofpromoting the differentiation of PDX1-negative definitive endoderm cellsto PDX1-positive foregut endoderm cells by determining the expression ofPDX1, HOXA13 and HOXC6 both before and after contacting the cells of thecell culture or cell population with the candidate differentiationfactor. In preferred embodiments, the expression of PDX1, HOXA13 and/orHOXC6 is determined Q-PCR.

It will be appreciated that in some embodiments, the expression of oneor more of PDX1, HOXA13 and HOXC6 can be determined at the time ofcontacting or shortly after contacting the cells of the cell cultures orcell populations with a candidate differentiation factor rather thanprior to contacting the cells with a candidate differentiation factor.In such embodiments, the expression of one or more of PDX1, HOXA13 andHOXC6 at the time of contacting or shortly after contacting the cellswith a candidate differentiation factor is compared to the expression ofone or more of PDX1, HOXA13 and HOXC6 at one or more times aftercontacting the cells with a candidate differentiation factor.

In some embodiments of the above-described methods, the one or moretimes at which PDX1 expression is determined after contacting the cellswith the candidate differentiation factor can range from about 1 hour toabout 10 days. For example, PDX1 expression can be determined about 1hour after contacting the cells with the candidate differentiationfactor, about 2 hours after contacting the cells with the candidatedifferentiation factor, about 4 hours after contacting the cells withthe candidate differentiation factor, about 6 hours after contacting thecells with the candidate differentiation factor, about 8 hours aftercontacting the cells with the candidate differentiation factor, about 10hours after contacting the cells with the candidate differentiationfactor, about 12 hours after contacting the cells with the candidatedifferentiation factor, about 16 hours after contacting the cells withthe candidate differentiation factor, about 24 hours after contactingthe cells with the candidate differentiation factor, about 2 days aftercontacting the cells with the candidate differentiation factor, about 3days after contacting the cells with the candidate differentiationfactor, about 4 days after contacting the cells with the candidatedifferentiation factor, about 5 days after contacting the cells with thecandidate differentiation factor, about 6 days after contacting thecells with the candidate differentiation factor, about 7 days aftercontacting the cells with the candidate differentiation factor, about 8days after contacting the cells with the candidate differentiationfactor, about 9 days after contacting the cells with the candidatedifferentiation factor, about 10 days after contacting the cells withthe candidate differentiation factor or more than 10 days aftercontacting the cells with the candidate differentiation factor.

Candidate differentiation factors for use in the methods describedherein can be selected from compounds, such as polypeptides and smallmolecules. For example, candidate polypeptides can include, but are notlimited to, growth factors, cytokines, chemokines, extracellular matrixproteins, and synthetic peptides. In a preferred embodiment, the growthfactor is from the FGF family, for example FGF-10. Candidate smallmolecules include, but are not limited to, compounds generated fromcombinatorial chemical synthesis and natural products, such as steroids,isoprenoids, terpenoids, phenylpropanoids, alkaloids and flavinoids. Itwill be appreciated by those of ordinary skill in the art that thousandsof classes of natural and synthetic small molecules are available andthat the small molecules contemplated for use in the methods describedherein are not limited to the classes exemplified above. Typically,small molecules will have a molecular weight less than 10,000 amu. In apreferred embodiment, the small molecule is a retinoid, for example RA.

Identification of Factors Capable of Promoting the Differentiation ofPDX1-Positive Foregut Endoderm Cells

Other aspects of the screening methods described herein relate tomethods of identifying one or more differentiation factors capable ofpromoting the differentiation of PDX1-positive foregut endoderm cells.In such methods, a cell culture or cell population comprisingPDX1-positive foregut endoderm cells is obtained and the expression of amarker in the cell culture or cell population is determined. Afterdetermining the expression of the marker, the cells of the cell cultureor cell population are contacted with a candidate differentiationfactor. In some embodiments, the expression of the marker is determinedat the time of contacting or shortly after contacting the cells with acandidate differentiation factor. The expression of the same marker isthen determined at one or more times after contacting the cells with thecandidate differentiation factor. If the expression of the marker hasincreased or decreased after contact with the candidate differentiationfactor as compared to the marker expression prior to contact with thecandidate differentiation factor, the candidate differentiation factoris identified as capable of promoting the differentiation ofPDX1-positive foregut endoderm cells. In preferred embodiments,expression of the marker is determined by Q-PCR.

In some embodiments of the above-described methods, the one or moretimes at which the marker expression is determined after contacting thecells with the candidate differentiation factor can range from about 1hour to about 10 days. For example, marker expression can be determinedabout 1 hour after contacting the cells with the candidatedifferentiation factor, about 2 hours after contacting the cells withthe candidate differentiation factor, about 4 hours after contacting thecells with the candidate differentiation factor, about 6 hours aftercontacting the cells with the candidate differentiation factor, about 8hours after contacting the cells with the candidate differentiationfactor, about 10 hours after contacting the cells with the candidatedifferentiation factor, about 12 hours after contacting the cells withthe candidate differentiation factor, about 16 hours after contactingthe cells with the candidate differentiation factor, about 24 hoursafter contacting the cells with the candidate differentiation factor,about 2 days after contacting the cells with the candidatedifferentiation factor, about 3 days after contacting the cells with thecandidate differentiation factor, about 4 days after contacting thecells with the candidate differentiation factor, about 5 days aftercontacting the cells with the candidate differentiation factor, about 6days after contacting the cells with the candidate differentiationfactor, about 7 days after contacting the cells with the candidatedifferentiation factor, about 8 days after contacting the cells with thecandidate differentiation factor, about 9 days after contacting thecells with the candidate differentiation factor, about 10 days aftercontacting the cells with the candidate differentiation factor or morethan 10 days after contacting the cells with the candidatedifferentiation factor.

As described previously, candidate differentiation factors for use inthe methods described herein can be selected from compounds such aspolypeptides and small molecules.

Although each of the methods disclosed herein have been described withrespect to PDX1-positive foregut endoderm cells, it will be appreciatedthat in certain embodiments, these methods can be used to producecompositions comprising the PDX1-positive foregut/midgut endoderm cellsthat are described herein and/or the PDX1-positive endoderm cells of theposterior portion of the foregut that are described herein. Furthermore,any of the PDX1-positive endoderm cell types disclosed in thisspecification can be utilized in the screening methods described herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting.

EXAMPLES

Many of the examples below describe the use of pluripotent human cells.Methods of producing pluripotent human cells are well known in the artand have been described numerous scientific publications, including U.S.Pat. Nos. 5,453,357, 5,670,372, 5,690,926, 6,090,622, 6,200,806 and6,251,671 as well as U.S. Patent Application Publication No.2004/0229350, the disclosures of which are incorporated herein byreference in their entireties.

Example 1 Human ES cells

For our studies of endoderm development we employed human embryonic stemcells, which are pluripotent and can divide seemingly indefinitely inculture while maintaining a normal karyotype. ES cells were derived fromthe 5-day-old embryo inner cell mass using either immunological ormechanical methods for isolation. In particular, the human embryonicstem cell line hESCyt-25 was derived from a supernumerary frozen embryofrom an in vitro fertilization cycle following informed consent by thepatient. Upon thawing the hatched blastocyst was plated on mouseembryonic fibroblasts (MEF), in ES medium (DMEM, 20% FBS, non essentialamino acids, beta-mercaptoethanol, ITS supplement). The embryo adheredto the culture dish and after approximately two weeks, regions ofundifferentiated hESCs were transferred to new dishes with MEFs.Transfer was accomplished with mechanical cutting and a brief digestionwith dispase, followed by mechanical removal of the cell clusters,washing and re-plating. Since derivation, hESCyt-25 has been seriallypassaged over 100 times. We employed the hESCyt-25 human embryonic stemcell line as our starting material for the production of definitiveendoderm.

It will be appreciated by those of skill in the art that stem cells orother pluripotent cells can also be used as starting material for thedifferentiation procedures described herein. For example, cells obtainedfrom embryonic gonadal ridges, which can be isolated by methods known inthe art, can be used as pluripotent cellular starting material.

Example 2 hESC3t-25 Characterization

The human embryonic stem cell line, hESCyt-25 has maintained a normalmorphology, karyotype, growth and self-renewal properties over 18 monthsin culture. This cell line displays strong immunoreactivity for theOCT4, SSEA-4 and TRA-1-60 antigens, all of which, are characteristic ofundifferentiated hESCs and displays alkaline phosphatase activity aswell as a morphology identical to other established hESC lines.Furthermore, the human stem cell line, hESCyt-25, also readily formsembryoid bodies (EBs) when cultured in suspension. As a demonstration ofits pluripotent nature, hESCyT-25 differentiates into various cell typesthat represent the three principal germ layers. Ectoderm production wasdemonstrated by Q-PCR for ZIC1 as well as immunocytochemistry (ICC) fornestin and more mature neuronal markers. Immunocytochemical staining forβ-III tubulin was observed in clusters of elongated cells,characteristic of early neurons. Previously, we treated EBs insuspension with retinoic acid, to induce differentiation of pluripotentstem cells to visceral endoderm (VE), an extra-embryonic lineage.Treated cells expressed high levels of Q-fetoprotein (AFP) and SOX7, twomarkers of VE, by 54 hours of treatment. Cells differentiated inmonolayer expressed AFP in sporadic patches as demonstrated byimmunocytochemical staining. As will be described below, the hESCyT-25cell line was also capable of forming definitive endoderm, as validatedby real-time quantitative polymerase chain reaction (Q-PCR) andimmunocytochemistry for SOX17, in the absence of AFP expression. Todemonstrate differentiation to mesoderm, differentiating EBs wereanalyzed for Brachyury gene expression at several time points. Brachyuryexpression increased progressively over the course of the experiment. Inview of the foregoing, the hESCyT-25 line is pluripotent as shown by theability to form cells representing the three germ layers.

Example 3 Production of SOX17 Antibody

A primary obstacle to the identification of definitive endoderm in hESCcultures is the lack of appropriate tools. We therefore undertook theproduction of an antibody raised against human SOX17 protein.

The marker SOX17 is expressed throughout the definitive endoderm as itforms during gastrulation and its expression is maintained in the guttube (although levels of expression vary along the A-P axis) untilaround the onset of organogenesis. SOX17 is also expressed in a subsetof extra-embryonic endoderm cells. No expression of this protein hasbeen observed in mesoderm or ectoderm. It has now been discovered thatSOX17 is an appropriate marker for the definitive endoderm lineage whenused in conjunction with markers to exclude extra-embryonic lineages.

As described in detail herein, the SOX17 antibody was utilized tospecifically examine effects of various treatments and differentiationprocedures aimed at the production of SOX17 positive definitive endodermcells. Other antibodies reactive to AFP, SPARC and Thrombomodulin werealso employed to rule out the production of visceral and parietalendoderm (extra-embryonic endoderm).

In order to produce an antibody against SOX17, a portion of the humanSOX17 cDNA (SEQ ID NO: 1) corresponding to amino acids 172-414 (SEQ IDNO: 2) in the carboxyterminal end of the SOX17 protein (FIG. 2) was usedfor genetic immunization in rats at the antibody production company,GENOVAC (Freiberg, Germany), according to procedures developed there.Procedures for genetic immunization can be found in U.S. Pat. Nos.5,830,876, 5,817,637, 6,165,993 and 6,261,281 as well as InternationalPatent Application Publication Nos. WO00/29442 and WO99/13915, thedisclosures of which are incorporated herein by reference in theirentireties.

Other suitable methods for genetic immunization are also described inthe non-patent literature. For example, Barry et al. describe theproduction of monoclonal antibodies by genetic immunization inBiotechniques 16: 616-620, 1994, the disclosure of which is incorporatedherein by reference in its entirety. Specific examples of geneticimmunization methods to produce antibodies against specific proteins canbe found, for example, in Costaglia et al., (1998) Genetic immunizationagainst the human thyrotropin receptor causes thyroiditis and allowsproduction of monoclonal antibodies recognizing the native receptor, J.Immunol. 160: 1458-1465; Kilpatrick et al (1998) Gene gun deliveredDNA-based immunizations mediate rapid production of murine monoclonalantibodies to the Flt-3 receptor, Hybridoma 17: 569-576; Schmolke etal., (1998) Identification of hepatitis G virus particles in human serumby E2-specific monoclonal antibodies generated by DNA immunization, J.Virol. 72: 4541-4545; Krasemann et al., (1999) Generation of monoclonalantibodies against proteins with an unconventional nucleic acid-basedimmunization strategy, J. Biotechnol. 73: 119-129; and Ulivieri et al.,(1996) Generation of a monoclonal antibody to a defined portion of theHeliobacter pylori vacuolating cytotoxin by DNA immunization, J.Biotechnol. 51: 191-194, the disclosures of which are incorporatedherein by reference in their entireties.

SOX7 and SOX18 are the closest Sox family relatives to SOX17 as depictedin the relational dendrogram shown in FIG. 3. We employed the human SOX7polypeptide as a negative control to demonstrate that the SOX17 antibodyproduced by genetic immunization is specific for SOX17 and does notreact with its closest family member. In particular, SOX7 and otherproteins were expressed in human fibroblasts, and then, analyzed forcross reactivity with the SOX17 antibody by Western blot and ICC. Forexample, the following methods were utilized for the production of theSOX17, SOX7 and EGFP expression vectors, their transfection into humanfibroblasts and analysis by Western blot. Expression vectors employedfor the production of SOX17, SOX7, and EGFP were pCMV6 (OriGeneTechnologies, Inc., Rockville, Md.), pCMV-SPORT6 (Invitrogen, Carlsbad,Calif.) and pEGFP-N 1 (Clonetech, Palo Alto, Calif.), respectively. Forprotein production, telomerase immortalized MDX human fibroblasts weretransiently transfected with supercoiled DNA in the presence ofLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Total cellularlysates were collected 36 hours post-transfection in 50 mM TRIS-HCl (pH8), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, containing a cocktail ofprotease inhibitors (Roche Diagnostics Corporation, Indianapolis, Ind.).Western blot analysis of 100 μg of cellular proteins, separated bySDS-PAGE on NuPAGE (4-12% gradient polyacrylamide, Invitrogen, Carlsbad,Calif.), and transferred by electro-blotting onto PDVF membranes(Hercules, Calif.), were probed with a 1/1000 dilution of the rat SOX17anti-serum in 10 mM TRIS-HCl (pH 8), 150 mM NaCl, 10% BSA, 0.05%Tween-20 (Sigma, St. Louis, Mo.), followed by Alkaline Phosphataseconjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, WestGrove, Pa.), and revealed through Vector Black Alkaline Phosphatasestaining (Vector Laboratories, Burlingame, Calif.). The proteins sizestandard used was wide range color markers (Sigma, St. Louis, Mo.).

In FIG. 4, protein extracts made from human fibroblast cells that weretransiently transfected with SOX17, SOX7 or EGFP cDNA's were probed onWestern blots with the SOX17 antibody. Only the protein extract fromhSOX17 transfected cells produced a band of 51 Kda which closely matchedthe predicted 46 Kda molecular weight of the human SOX17 protein. Therewas no reactivity of the SOX17 antibody to extracts made from eitherhuman SOX7 or EGFP transfected cells. Furthermore, the SOX17 antibodyclearly labeled the nuclei of human fibroblast cells transfected withthe hSOX17 expression construct but did not label cells transfected withEGFP alone. As such, the SOX17 antibody exhibits specificity by ICC.

Example 4 Validation of SOX17 Antibody as a Marker of DefinitiveEndoderm

Partially differentiated hESCs were co-labeled with SOX17 and AFPantibodies to demonstrate that the SOX17 antibody is specific for humanSOX17 protein and furthermore marks definitive endoderm. It has beendemonstrated that SOX17, SOX7 (which is a closely related member of theSOX gene family subgroup F (FIG. 3)) and AFP are each expressed invisceral endoderm. However, AFP and SOX7 are not expressed in definitiveendoderm cells at levels detectable by ICC, and thus, they can beemployed as negative markers for bonifide definitive endoderm cells. Itwas shown that SOX17 antibody labels populations of cells that exist asdiscrete groupings of cells or are intermingled with AFP positive cells.In particular, FIG. 5A shows that small numbers of SOX17 cells wereco-labeled with AFP; however, regions were also found where there werelittle or no AFP⁺ cells in the field of SOX17⁺ cells (FIG. 5B).Similarly, since parietal endoderm has been reported to express SOX17,antibody co-labeling with SOX17 together with the parietal markers SPARCand/or Thrombomodulin (TM) can be used to identify the SOX17⁺ cells thatare parietal endoderm. As shown in FIGS. 6A-C, Thrombomodulin and SOX17co-labeled parietal endoderm cells were produced by randomdifferentiation of hES cells.

In view of the above cell labeling experiments, the identity of adefinitive endoderm cell can be established by the marker profileSOX17^(hi)/AFP^(lo)/[TM^(lo) or SPARC^(lo)]. In other words, theexpression of the SOX17 marker is greater than the expression of the AFPmarker, which is characteristic of visceral endoderm, and the TM orSPARC markers, which are characteristic of parietal endoderm.Accordingly, those cells positive for SOX17 but negative for AFP andnegative for TM or SPARC are definitive endoderm.

As a further evidence of the specificity of theSOX17^(hi)/AFP^(lo)/TM^(lo)/SPARC^(lo) marker profile as predictive ofdefinitive endoderm, SOX17 and AFP gene expression was quantitativelycompared to the relative number of antibody labeled cells. As shown inFIG. 7A, hESCs treated with retinoic acid (visceral endoderm inducer),or activin A (definitive endoderm inducer), resulted in a 10-folddifference in the level of SOX17 mRNA expression. This result mirroredthe 10-fold difference in SOX17 antibody-labeled cell number (FIG. 7B).Furthermore, as shown in FIG. 8A, activin A treatment of hESCssuppressed AFP gene expression by 6.8-fold in comparison to notreatment. This was visually reflected by a dramatic decrease in thenumber of AFP labeled cells in these cultures as shown in FIGS. 8B-C. Toquantify this further, it was demonstrated that this approximately7-fold decrease in AFP gene expression was the result of a similar7-fold decrease in AFP antibody-labeled cell number as measured by flowcytometry (FIGS. 9A-B). This result is extremely significant in that itindicates that quantitative changes in gene expression as seen by Q-PCRmirror changes in cell type specification as observed by antibodystaining.

Incubation of hESCs in the presence of Nodal family members (Nodal,activin A and activin B—NAA) resulted in a significant increase in SOX17antibody-labeled cells over time. By 5 days of continuous activintreatment greater than 50% of the cells were labeled with SOX17 (FIGS.10A-F). There were few or no cells labeled with AFP after 5 days ofactivin treatment.

In summary, the antibody produced against the carboxy-terminal 242 aminoacids of the human SOX17 protein identified human SOX17 protein onWestern blots but did not recognize SOX7, it's closest Sox familyrelative. The SOX17 antibody recognized a subset of cells indifferentiating hESC cultures that were primarily SOX17⁺/AFP^(lo/−)(greater than 95% of labeled cells) as well as a small percentage (<5%)of cells that co-label for SOX17 and AFP (visceral endoderm). Treatmentof hESC cultures with activins resulted in a marked elevation of SOX17gene expression as well as SOX17 labeled cells and dramaticallysuppressed the expression of AFP mRNA and the number of cells labeledwith AFP antibody.

Example 5 Q-PCR Gene Expression Assay

In the following experiments, real-time quantitative RT-PCR (Q-PCR) wasthe primary assay used for screening the effects of various treatmentson hESC differentiation. In particular, real-time measurements of geneexpression were analyzed for multiple marker genes at multiple timepoints by Q-PCR. Marker genes characteristic of the desired as well asundesired cell types were evaluated to gain a better understanding ofthe overall dynamics of the cellular populations. The strength of Q-PCRanalysis includes its extreme sensitivity and relative ease ofdeveloping the necessary markers, as the genome sequence is readilyavailable. Furthermore, the extremely high sensitivity of Q-PCR permitsdetection of gene expression from a relatively small number of cellswithin a much larger population. In addition, the ability to detect verylow levels of gene expression provides indications for “differentiationbias” within the population. The bias towards a particulardifferentiation pathway, prior to the overt differentiation of thosecellular phenotypes, is unrecognizable using immunocytochemicaltechniques. For this reason, Q-PCR provides a method of analysis that isat least complementary and potentially much superior toimmunocytochemical techniques for screening the success ofdifferentiation treatments. Additionally, Q-PCR provides a mechanism bywhich to evaluate the success of a differentiation protocol in aquantitative format at semi-high throughput scales of analysis.

The approach taken here was to perform relative quantitation using SYBRGreen chemistry on a Rotor Gene 3000 instrument (Corbett Research) and atwo-step RT-PCR format. Such an approach allowed for the banking of cDNAsamples for analysis of additional marker genes in the future, thusavoiding variability in the reverse transcription efficiency betweensamples.

Primers were designed to lie over exon-exon boundaries or span intronsof at least 800 bp when possible, as this has been empiricallydetermined to eliminate amplification from contaminating genomic DNA.When marker genes were employed that do not contain introns or theypossess pseudogenes, DNase I treatment of RNA samples was performed.

We routinely used Q-PCR to measure the gene expression of multiplemarkers of target and non-target cell types in order to provide a broadprofile description of gene expression in cell samples. The markersrelevant for the early phases of hESC differentiation (specificallyectoderm, mesoderm, definitive endoderm and extra-embryonic endoderm)and for which validated primer sets are available are provided below inTable 1. The human specificity of these primer sets has also beendemonstrated. This is an important fact since the hESCs were often grownon mouse feeder layers. Most typically, triplicate samples were takenfor each condition and independently analyzed in duplicate to assess thebiological variability associated with each quantitative determination.

To generate PCR template, total RNA was isolated using RNeasy (Qiagen)and quantitated using RiboGreen (Molecular Probes). Reversetranscription from 350-500 ng of total RNA was carried out using theiScript reverse transcriptase kit (BioRad), which contains a mix ofoligo-dT and random primers. Each 20 μL reaction was subsequentlydiluted up to 100 μL total volume and 3 μL was used in each 10 μL Q-PCRreaction containing 400 nM forward and reverse primers and 5 μL 2X SYBRGreen master mix (Qiagen). Two step cycling parameters were usedemploying a 5 second denature at 85-94° C. (specifically selectedaccording to the melting temp of the amplicon for each primer set)followed by a 45 second anneal/extend at 60° C. Fluorescence data wascollected during the last 15 seconds of each extension phase. A threepoint, 10-fold dilution series was used to generate the standard curvefor each run and cycle thresholds (Ct's) were converted to quantitativevalues based on this standard curve. The quantitated values for eachsample were normalized to housekeeping gene performance and then averageand standard deviations were calculated for triplicate samples. At theconclusion of PCR cycling, a melt curve analysis was performed toascertain the specificity of the reaction. A single specific product wasindicated by a single peak at the T_(m) appropriate for that PCRamplicon. In addition, reactions performed without reverse transcriptaseserved as the negative control and do not amplify.

A first step in establishing the Q-PCR methodology was validation ofappropriate housekeeping genes (HGs) in the experimental system. Sincethe HG was used to normalize across samples for the RNA input, RNAintegrity and RT efficiency, it was of value that the HG exhibited aconstant level of expression over time in all sample types in order forthe normalization to be meaningful. We measured the expression levels ofCyclophilin G, hypoxanthine phosphoribosyltransferase 1 (HPRT),beta-2-microglobulin, hydroxymethylbiane synthase (HMBS), TATA-bindingprotein (TBP), and glucoronidase beta (GUS) in differentiating hESCs.Our results indicated that beta-2-microglobulin expression levelsincreased over the course of differentiation and therefore we excludedthe use of this gene for normalization. The other genes exhibitedconsistent expression levels over time as well as across treatments. Weroutinely used both Cyclophilin G and GUS to calculate a normalizationfactor for all samples. The use of multiple HGs simultaneously reducesthe variability inherent to the normalization process and increases thereliability of the relative gene expression values.

After obtaining genes for use in normalization, Q-PCR was then utilizedto determine the relative gene expression levels of many marker genesacross samples receiving different experimental treatments. The markergenes employed have been chosen because they exhibit enrichment inspecific populations representative of the early germ layers and inparticular have focused on sets of genes that are differentiallyexpressed in definitive endoderm and extra-embryonic endoderm. Thesegenes as well as their relative enrichment profiles are highlighted inTable 1.

TABLE 1 Germ Layer Gene Expression Domains Endoderm SOX17 definitive,visceral and parietal endoderm MIXL1 endoderm and mesoderm GATA4definitive and primitive endoderm HNF3b definitive endoderm andprimitive endoderm, mesoderm, neural plate GSC endoderm and mesodermExtra- SOX7 visceral endoderm embryonic AFP visceral endoderm, liverSPARC parietal endoderm TM parietal endoderm/trophectoderm Ectoderm ZIC1neural tube, neural progenitors Mesoderm BRACH nascent mesoderm

Since many genes are expressed in more than one germ layer it is usefulto quantitatively compare expression levels of many genes within thesame experiment. SOX17 is expressed in definitive endoderm and to asmaller extent in visceral and parietal endoderm. SOX7 and AFP areexpressed in visceral endoderm at this early developmental time point.SPARC and TM are expressed in parietal endoderm and Brachyury isexpressed in early mesoderm.

Definitive endoderm cells were predicted to express high levels of SOX17mRNA and low levels of AFP and SOX7 (visceral endoderm), SPARC (parietalendoderm) and Brachyury (mesoderm). In addition, ZIC1 was used here tofurther rule out induction of early ectoderm. Finally, GATA4 and HNF3bwere expressed in both definitive and extra-embryonic endoderm, andthus, correlate with SOX17 expression in definitive endoderm (Table 1).A representative experiment is shown in FIGS. 11-14 which demonstrateshow the marker genes described in Table 1 correlate with each otheramong the various samples, thus highlighting specific patterns ofdifferentiation to definitive endoderm and extra-embryonic endoderm aswell as to mesodermal and neural cell types.

In view of the above data it is clear that increasing doses of activinresulted in increasing SOX17 gene expression. Further this SOX17expression predominantly represented definitive endoderm as opposed toextra-embryonic endoderm. This conclusion stems from the observationthat SOX17 gene expression was inversely correlated with AFP, SOX7, andSPARC gene expression.

Example 6 Directed Differentiation of Human ES Cells to DefinitiveEndoderm

Human ES cell cultures randomly differentiate if cultured underconditions that do not actively maintain their undifferentiated state.This heterogeneous differentiation results in production ofextra-embryonic endoderm cells comprised of both parietal and visceralendoderm (AFP, SPARC and SOX7 expression) as well as early ectodermaland mesodermal derivatives as marked by ZIC1 and Nestin (ectoderm) andBrachyury (mesoderm) expression. Definitive endoderm cell appearance hasnot been examined or specified for lack of specific antibody markers inES cell cultures. As such, and by default, early definitive endodermproduction in ES cell cultures has not been well studied. Sincesatisfactory antibody reagents for definitive endoderm cells have beenunavailable, most of the characterization has focused on ectoderm andextra-embryonic endoderm. Overall, there are significantly greaternumbers of extra-embryonic and neurectodermal cell types in comparisonto SOX17^(hi) definitive endoderm cells in randomly differentiated EScell cultures.

As undifferentiated hESC colonies expand on a bed of fibroblast feeders,the cells at the edges of the colony take on alternative morphologiesthat are distinct from those cells residing within the interior of thecolony. Many of these outer edge cells can be distinguished by theirless uniform, larger cell body morphology and by the expression ofhigher levels of OCT4. It has been described that as ES cells begin todifferentiate they alter the levels of OCT4 expression up or downrelative to undifferentiated ES cells. Alteration of OCT4 levels aboveor below the undifferentiated threshold may signify the initial stagesof differentiation away from the pluripotent state.

When undifferentiated colonies were examined by SOX17immunocytochemistry, occasionally small 10-15-cell clusters ofSOX17-positive cells were detected at random locations on the peripheryand at the junctions between undifferentiated hESC colonies. As notedabove, these scattered pockets of outer colony edges appeared to be someof the first cells to differentiate away from the classical ES cellmorphology as the colony expanded in size and became more crowded.Younger, smaller fully undifferentiated colonies (<1 mm; 4-5 days old)showed no SOX17 positive cells within or at the edges of the colonieswhile older, larger colonies (1-2 mm diameter, >5 days old) had sporadicisolated patches of SOX17 positive, AFP negative cells at the peripheryof some colonies or in regions interior to the edge that did not displaythe classical hESC morphology described previously. Given that this wasthe first development of an effective SOX17 antibody, definitiveendoderm cells generated in such early “undifferentiated” ES cellcultures have never been previously demonstrated.

Based on negative correlations of SOX17 and SPARC gene expression levelsby Q-PCR, the vast majority of these SOX17 positive, AFP negative cellswill be negative for parietal endoderm markers by antibody co-labeling.This was specifically demonstrated for TM-expressing parietal endodermcells as shown in FIGS. 15A-B. Exposure to Nodal factors activin A and Bresulted in a dramatic decrease in the intensity of TM expression andthe number of TM positive cells. By triple labeling using SOX17, AFP andTM antibodies on an activin treated culture, clusters of SOX17 positivecells that were also negative for AFP and TM were observed (FIGS.16A-D). These are the first cellular demonstrations of SOX17 positivedefinitive endoderm cells in differentiating hESC cultures (FIGS. 16A-Dand 17).

With the SOX17 antibody and Q-PCR tools described above we have exploreda number of procedures capable of efficiently programming hESCs tobecome SOX17^(hi)/AFP^(lo)/SPARC/TM^(lo) definitive endoderm cells. Weapplied a variety of differentiation protocols aimed at increasing thenumber and proliferative capacity of these cells as measured at thepopulation level by Q-PCR for SOX17 gene expression and at the level ofindividual cells by antibody labeling of SOX17 protein.

We were the first to analyze and describe the effect of TGFβ familygrowth factors, such as Nodal/activin/BMP, for use in creatingdefinitive endoderm cells from embryonic stem cells in in vitro cellcultures. In typical experiments, activin A, activin B, BMP orcombinations of these growth factors were added to cultures ofundifferentiated human stem cell line hESCyt-25 to begin thedifferentiation process.

As shown in FIG. 19, addition of activin A at 100 ng/ml resulted in a19-fold induction of SOX17 gene expression vs. undifferentiated hESCs byday 4 of differentiation. Adding activin B, a second member of theactivin family, together with activin A, resulted in a 37-fold inductionover undifferentiated hESCs by day 4 of combined activin treatment.Finally, adding a third member of the TGFβ family from the Nodal/Activinand BMP subgroups, BMP4, together with activin A and activin B,increased the fold induction to 57 times that of undifferentiated hESCs(FIG. 19). When SOX17 induction with activins and BMP was compared to nofactor medium controls 5-, 10-, and 15-fold inductions resulted at the4-day time point. By five days of triple treatment with activins A, Band BMP, SOX17 was induced more than 70 times higher than hESCs. Thesedata indicate that higher doses and longer treatment times of theNodal/activin TGFβ family members results in increased expression ofSOX17.

Nodal and related molecules activin A, B and BMP facilitate theexpression of SOX17 and definitive endoderm formation in vivo or invitro. Furthermore, addition of BMP results in an improved SOX17induction possibly through the further induction of Cripto, the Nodalco-receptor.

We have demonstrated that the combination of activins A and B togetherwith BMP4 result in additive increases in SOX17 induction and hencedefinitive endoderm formation. BMP4 addition for prolonged periods (>4days), in combination with activin A and B may induce SOX17 in parietaland visceral endoderm as well as definitive endoderm. In someembodiments of the present invention, it is therefore valuable to removeBMP4 from the treatment within 4 days of addition.

To determine the effect of TGFβ factor treatment at the individual celllevel, a time course of TGFβ factor addition was examined using SOX17antibody labeling. As previously shown in FIGS. 10A-F, there was adramatic increase in the relative number of SOX17 labeled cells overtime. The relative quantification (FIG. 20) shows more than a 20-foldincrease in SOX17-labeled cells. This result indicates that both thenumbers of cells as well SOX17 gene expression level are increasing withtime of TGFβ factor exposure. As shown in FIG. 21, after four days ofexposure to Nodal, activin A, activin B and BMP4, the level of SOX17induction reached 168-fold over undifferentiated hESCs. FIG. 22 showsthat the relative number of SOX17-positive cells was also doseresponsive activin A doses of 100 ng/ml or more were capable of potentlyinducing SOX17 gene expression and cell number.

In addition to the TGFβ family members, the Wnt family of molecules mayplay a role in specification and/or maintenance of definitive endoderm.The use of Wnt molecules was also beneficial for the differentiation ofhESCs to definitive endoderm as indicated by the increased SOX17 geneexpression in samples that were treated with activins plus Wnt3a overthat of activins alone (FIG. 23).

All of the experiments described above were performed using a tissueculture medium containing 10% serum with added factors. Surprisingly, wediscovered that the concentration of serum had an effect on the level ofSOX17 expression in the presence of added activins as shown in FIGS.24A-C. When serum levels were reduced from 10% to 2%, SOX17 expressiontripled in the presence of activins A and B.

Finally, we demonstrated that activin induced SOX17⁺ cells divide inculture as depicted in FIGS. 25A-D. The arrows show cells labeled withSOX17/PCNA/DAPI that are in mitosis as evidenced by thePCNA/DAPI-labeled mitotic plate pattern and the phase contrast mitoticprofile.

Example 7 Chemokine Receptor 4 (CXCR4) Expression Correlates withMarkers for Definitive Endoderm and not Markers for Mesoderm, Ectodermor Visceral Endoderm

As described above, hESCs can be induced to differentiate to thedefinitive endoderm germ layer by the application of cytokines of theTGFβ family and more specifically of the activin/nodal subfamily.Additionally, we have shown that the proportion of fetal bovine serum(FBS) in the differentiation culture medium effects the efficiency ofdefinitive endoderm differentiation from hESCs. This effect is such thatat a given concentration of activin A in the medium, higher levels ofFBS will inhibit maximal differentiation to definitive endoderm. In theabsence of exogenous activin A, differentiation of hESCs to thedefinitive endoderm lineage is very inefficient and the FBSconcentration has much milder effects on the differentiation process ofhESCs.

In these experiments, hESCs were differentiated by growing in RPMImedium (Invitrogen, Carlsbad, Calif.; cat# 61870-036) supplemented with0.5%, 2.0% or 10% FBS and either with or without 100 ng/ml activin A for6 days. In addition, a gradient of FBS ranging from 0.5% to 2.0% overthe first three days of differentiation was also used in conjunctionwith 100 ng/ml of activin A. After the 6 days, replicate samples werecollected from each culture condition and analyzed for relative geneexpression by real-time quantitative PCR. The remaining cells were fixedfor immunofluorescent detection of SOX17 protein.

The expression levels of CXCR4 varied dramatically across the 7 cultureconditions used (FIG. 26). In general, CXCR4 expression was high inactivin A treated cultures (A100) and low in those which did not receiveexogenous activin A (NF). In addition, among the A100 treated cultures,CXCR4 expression was highest when FBS concentration was lowest. Therewas a remarkable decrease in CXCR4 level in the 10% FBS condition suchthat the relative expression was more in line with the conditions thatdid not receive activin A (NF).

As described above, expression of the SOX17, GSC, MIXL1, and HNF3 pgenes is consistent with the characterization of a cell as definitiveendoderm. The relative expression of these four genes across the 7differentiation conditions mirrors that of CXCR4 (FIGS. 27A-D). Thisdemonstrates that CXCR4 is also a marker of definitive endoderm.

Ectoderm and mesoderm lineages can be distinguished from definitiveendoderm by their expression of various markers. Early mesodermexpresses the genes Brachyury and MOX1 while nascent neuro-ectodermexpresses SOX1 and ZIC1. FIGS. 28A-D demonstrate that the cultures whichdid not receive exogenous activin A were preferentially enriched formesoderm and ectoderm gene expression and that among the activin Atreated cultures, the 10% FBS condition also had increased levels ofmesoderm and ectoderm marker expression. These patterns of expressionwere inverse to that of CXCR4 and indicated that CXCR4 was not highlyexpressed in mesoderm or ectoderm derived from hESCs at thisdevelopmental time period.

Early during mammalian development, differentiation to extra-embryoniclineages also occurs. Of particular relevance here is thedifferentiation of visceral endoderm that shares the expression of manygenes in common with definitive endoderm, including SOX17. Todistinguish definitive endoderm from extra-embryonic visceral endodermone should examine a marker that is distinct between these two. SOX7represents a marker that is expressed in the visceral endoderm but notin the definitive endoderm lineage. Thus, culture conditions thatexhibit robust SOX17 gene expression in the absence of SOX7 expressionare likely to contain definitive and not visceral endoderm. It is shownin FIG. 28E that SOX7 was highly expressed in cultures that did notreceive activin A, SOX7 also exhibited increased expression even in thepresence of activin A when FBS was included at 10%. This pattern is theinverse of the CXCR4 expression pattern and suggests that CXCR4 is nothighly expressed in visceral endoderm.

The relative number of SOX17 immunoreactive (SOX17⁺) cells present ineach of the differentiation conditions mentioned above was alsodetermined. When hESCs were differentiated in the presence of high doseactivin A and low FBS concentration (0.5% -2.0%) SOX17⁺ cells wereubiquitously distributed throughout the culture. When high dose activinA was used but FBS was included at 10% (v/v), the SOX17⁺ cells appearedat much lower frequency and always appeared in isolated clusters ratherthan evenly distributed throughout the culture (FIGS. 29A and C as wellas B and E). A further decrease in SOX17⁺ cells was seen when noexogenous activin A was used. Under these conditions the SOX17⁺ cellsalso appeared in clusters and these clusters were smaller and much morerare than those found in the high activin A, low FBS treatment (FIG. 29C and F). These results demonstrate that the CXCR4 expression patternsnot only correspond to definitive endoderm gene expression but also tothe number of definitive endoderm cells in each condition.

Example 8 Differentiation Conditions that Enrich for Definitive EndodermIncrease the Proportion of CXCR4 Positive Cells

The dose of activin A also effects the efficiency at which definitiveendoderm can be derived from hESCs. This example demonstrates thatincreasing the dose of activin A increases the proportion of CXCR4⁺cells in the culture.

hESCs were differentiated in RPMI media supplemented with 0.5%-2% FBS(increased from 0.5% to 1.0% to 2.0% over the first 3 days ofdifferentiation) and either 0, 10, or 100 ng/ml of activin A. After 7days of differentiation the cells were dissociated in PBS withoutCa²⁺/Mg²⁺ containing 2% FBS and 2 mM (EDTA) for 5 minutes at roomtemperature. The cells were filtered through 35 μm nylon filters,counted and pelleted. Pellets were resuspended in a small volume of 50%human serum/50% normal donkey serum and incubated for 2 minutes on iceto block non-specific antibody binding sites. To this, 1 μl of mouseanti-CXCR4 antibody (Abeam, cat# ab10403-100) was added per 50 μl(containing approximately 10⁵ cells) and labeling proceeded for 45minutes on ice. Cells were washed by adding 5 ml of PBS containing 2%human serum (buffer) and pelleted. A second wash with 5 ml of buffer wascompleted then cells were resuspended in 50 μl buffer per 10⁵ cells.Secondary antibody (FITC conjugated donkey anti-mouse; JacksonImmunoResearch, cat# 715-096-151) was added at 5 μg/ml finalconcentration and allowed to label for 30 minutes followed by two washesin buffer as above. Cells were resuspended at 5×10⁶ cells/ml in bufferand analyzed and sorted using a FACS Vantage (Beckton Dickenson) by thestaff at the flow cytometry core facility (The Scripps ResearchInstitute). Cells were collected directly into RLT lysis buffer (Qiagen)for subsequent isolation of total RNA for gene expression analysis byreal-time quantitative PCR.

The number of CXCR4⁺ cells as determined by flow cytometry were observedto increase dramatically as the dose of activin A was increased in thedifferentiation culture media (FIGS. 30A-C). The CXCR4⁺cells were thosefalling within the R4 gate and this gate was set using a secondaryantibody-only control for which 0.2% of events were located in the R4gate. The dramatically increased numbers of CXCR4⁺ cells correlates witha robust increase in definitive endoderm gene expression as activin Adose is increased (FIGS. 31A-D).

Example 9 Isolation of CXCR4 Positive Cells Enriches for DefinitiveEndoderm Gene Expression and Depletes Cells Expressing Markers ofMesoderm Ectoderm and Visceral Endoderm

The CXCR4⁺and CXCR4 cells identified in Example 8 above were collectedand analyzed for relative gene expression and the gene expression of theparent populations was determined simultaneously.

The relative levels of CXCR4 gene expression was dramatically increasedwith increasing dose of activin A (FIG. 32). This correlated very wellwith the activin A dose-dependent increase of CXCR4⁺ cells (FIGS.30A-C). It is also clear that isolation of the CXCR4⁺ cells from eachpopulation accounted for nearly all of the CXCR4 gene expression in thatpopulation. This demonstrates the efficiency of the FACS method forcollecting these cells.

Gene expression analysis revealed that the CXCR4⁺ cells contain not onlythe majority of the CXCR4 gene expression, but they also contained geneexpression for other markers of definitive endoderm. As shown in FIGS. 31A-D, the CXCR4⁺ cells were further enriched over the parent A100population for SOX17, GSC, HNF3B, and MIXL1. In addition, the CXCR4⁻fraction contained very little gene expression for these definitiveendoderm markers. Moreover, the CXCR4⁺ and CXCR4⁻ populations displayedthe inverse pattern of gene expression for markers of mesoderm, ectodermand extra-embryonic endoderm. FIGS. 33A-D shows that the CXCR4⁺ cellswere depleted for gene expression of Brachyury, MOX1, ZIC1, and SOX7relative to the A100 parent population. This A100 parent population wasalready low in expression of these markers relative to the low dose orno activin A conditions. These results show that the isolation of CXCR4⁺cells from hESCs differentiated in the presence of high activin A yieldsa population that is highly enriched for and substantially puredefinitive endoderm.

Example 10 Quantitation of Definitive Endoderm Cells in a CellPopulation Using CXCR4

To confirm the quantitation of the proportion of definitive endodermcells present in a cell culture or cell population as determinedpreviously herein and as determined in U.S. Provisional PatentApplication No. 60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23,2003, the disclosure of which is incorporated herein by reference in itsentirety, cells expressing CXCR4 and other markers of definitiveendoderm were analyzed by FACS.

Using the methods such as those described in the above Examples, hESCswere differentiated to produce definitive endoderm. In particular, toincrease the yield and purity in differentiating cell cultures, theserum concentration of the medium was controlled as follows: 0.2% FBS onday 1, 1.0% FBS on day 2 and 2.0% FBS on days 3-6. Differentiatedcultures were sorted by FACS using three cell surface epitopes,E-Cadherin, CXCR4, and Thrombomodulin. Sorted cell populations were thenanalyzed by Q-PCR to determine relative expression levels of markers fordefinitive and extraembryonic-endoderm as well as other cell types.CXCR4 sorted cells taken from optimally differentiated cultures resultedin the isolation of definitive endoderm cells that were >98% pure.

Table 2 shows the results of a marker analysis for a definitive endodermculture that was differentiated from hESCs using the methods describedherein.

TABLE 2 Composition of Definitive Endoderm Cultures Percent PercentPercent Percent of Definitive Extraembryonic hES Marker(s) cultureEndoderm endoderm cells SOX17 70-80 100 Thrombomodulin <2 0 75 AFP <1 025 CXCR4 70-80 100 0 ECAD 10 0 100 other (ECAD neg.) 10-20 Total 100 100100 100

In particular, Table 2 indicates that CXCR4 and SOX17 positive cells(endoderm) comprised from 70%-80% of the cells in the cell culture. Ofthese SOX17-expressing cells, less than 2% expressed TM (parietalendoderm) and less than 1% expressed AFP (visceral endoderm). Aftersubtracting the proportion of TM-positive and AFP-positive cells(combined parietal and visceral endoderm; 3% total) from the proportionof SOX17/CXCR4 positive cells, it can be seen that about 67% to about77% of the cell culture was definitive endoderm. Approximately 10% ofthe cells were positive for E-Cadherin (ECAD), which is a marker forhESCs, and about 10-20% of the cells were of other cell types.

We have discovered that the purity of definitive endoderm in thedifferentiating cell cultures that are obtained prior to FACS separationcan be improved as compared to the above-described low serum procedureby maintaining the FBS concentration at ≦0.5% throughout the 5-6 daydifferentiation procedure. However, maintaining the cell culture at≦0.5% throughout the 5-6 day differentiation procedure also results in areduced number of total definitive endoderm cells that are produced.

Definitive endoderm cells produced by methods described herein have beenmaintained and expanded in culture in the presence of activin forgreater than 50 days without appreciable differentiation. In such cases,SOX17, CXCR4, MIXL1, GATA4, HNF3β expression is maintained over theculture period. Additionally, TM, SPARC, OCT4, AFP, SOX7, ZIC1 and BRACHwere not detected in these cultures. It is likely that such cells can bemaintained and expanded in culture for substantially longer than 50 dayswithout appreciable differentiation.

Example 11 Additional Markers of Definitive Endoderm Cells

In the following experiment, RNA was isolated from purified definitiveendoderm and human embryonic stem cell populations. Gene expression wasthen analyzed by gene chip analysis of the RNA from each purifiedpopulation. Q-PCR was also performed to further investigate thepotential of genes expressed in definitive endoderm, but not inembryonic stem cells, as a marker for definitive endoderm.

Human embryonic stem cells (hESCs) were maintained in DMEM/F12 mediasupplemented with 20% KnockOut Serum Replacement, 4 ng/ml recombinanthuman basic fibroblast growth factor (bFGF), 0.1 mM 2-mercaptoethanol,L-glutamine, non-essential amino acids and penicillin/streptomycin.hESCs were differentiated to definitive endoderm by culturing for 5 daysin RPMI media supplemented with 100 ng/ml of recombinant human activinA, fetal bovine serum (FBS), and penicillin/streptomycin. Theconcentration of FBS was varied each day as follows: 0.1% (first day),0.2% (second day), 2% (days 3-5).

Cells were isolated by fluorescence activated cell sorting (FACS) inorder to obtain purified populations of hESCs and definitive endodermfor gene expression analysis. Immuno-purification was achieved for hESCsusing SSEA4 antigen (R&D Systems, cat# FAB1435P) and for definitiveendoderm using CXCR4 (R&D Systems, cat# FAB170P). Cells were dissociatedusing trypsin/EDTA (Invitrogen, cat# 25300-054), washed in phosphatebuffered saline (PBS) containing 2% human serum and resuspended in 100%human serum on ice for 10 minutes to block non-specific binding.Staining was carried out for 30 minutes on ice by adding 200 μl ofphycoerythrin-conjugated antibody to 5×10⁶ cells in 800 μl human serum.Cells were washed twice with 8 ml of PBS buffer and resuspended in 1 mlof the same. FACS isolation was carried out by the core facility of TheScripps Research Institute using a FACS Vantage (BD Biosciences). Cellswere collected directly into RLT lysis buffer and RNA was isolated byRNeasy according to the manufacturers instructions (Qiagen).

Purified RNA was submitted in duplicate to Expression Analysis (Durham,N.C.) for generation of the expression profile data using the Affymetrixplatform and U133 Plus 2.0 high-density oligonucleotide arrays. Datapresented is a group comparison that identifies genes differentiallyexpressed between the two populations, hESCs and definitive endoderm.Genes that exhibited a robust upward change in expression level overthat found in hESCs were selected as new candidate markers that arehighly characteristic of definitive endoderm. Select genes were assayedby Q-PCR, as described above, to verify the gene expression changesfound on the gene chip and also to investigate the expression pattern ofthese genes during a time course of hESC differentiation.

FIGS. 34A-M show the gene expression results for certain markers.Results are displayed for cell cultures analyzed 1, 3 and 5 days afterthe addition of 100 ng/ml activin A, CXCR4-expressing definitiveendoderm cells purified at the end of the five day differentiationprocedure (CXDE), and in purified hESCs. A comparison of FIGS. 34C andG-M demonstrates that the six marker genes, FGF17, VWF, CALCR, FOXQ1,CMKOR1 and CRIP1, exhibit an expression pattern that is almost identicalto each other and which is also identical to the pattern of expressionof CXCR4 and the ratio of SOX17/SOX7. As described previously, SOX17 isexpressed in both the definitive endoderm as well as in theSOX7-expressing extra-embryonic endoderm. Since SOX7 is not expressed inthe definitive endoderm, the ratio of SOX17/SOX7 provides a reliableestimate of definitive endoderm contribution to the SOX17 expressionwitnessed in the population as a whole. The similarity of panels G-L andM to panel C indicates that FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1are likely markers of definitive endoderm and that they are notsignificantly expressed in extra-embryonic endoderm cells.

It will be appreciated that the Q-PCR results described herein can befurther confirmed by ICC.

Example 12 Retinoic Acid and FGF-10 Induces PDX1 Specifically inDefinitive Endoderm Cultures

The following experiment demonstrates that RA and FGF-10 induces theexpression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, 1 μM RA and 50 ng/ml FGF-10 were added to thecell culture. Forty-eight hours after the RA/FGF-10 addition, theexpression of the PDX1 marker gene and other marker genes not specificto foregut endoderm were quantitated by Q-PCR.

The application of RA to definitive endoderm cells caused a robustincrease in PDX1 gene expression (see FIG. 35) without increasing theexpression of visceral endoderm (SOX7, AFP), neural (SOX1, ZIC1), orneuronal (NFM) gene expression markers (see FIG. 36A-F). PDX1 geneexpression was induced to levels approximately 500-fold higher thanobserved in definitive endoderm after 48 hours exposure to 1 μM RA and50 ng/ml FGF-10. Furthermore, these results show that substantial PDX1induction occurred only in cell cultures which had been previouslydifferentiated to definitive endoderm (SOX17) as indicated by the160-fold higher PDX1 expression found in the activin treated cellcultures relative to those cultures that received no activin prior to RAapplication.

Example 13 FGF-10 Provides Additional Increase in PDX1 Expression OverRA Alone

This Example shows that the combination of RA and FGF-10 induces PDX1expression to a greater extent than RA alone.

As in the previous Example, hESCs were cultured with or without activinsfor four days. On day four, the cells were treated with one of thefollowing: 1 μM RA alone; 1 μM RA in combination with either FGF-4 orFGF-10; or 1 μM RA in combination with both FGF-4 and FGF-10. Theexpression of PDX1, SOX7 and NFM were quantitated by Q-PCR ninety sixhours after RA or RA/FGF.

The treatment of hESC cultures with activin followed by retinoic acidinduced a 60-fold increase in PDX1 gene expression. The addition ofFGF-4 to the RA treatment induced slightly more PDX1 (approximately3-fold over RA alone). However, by adding FGF-10 and retinoic acidtogether, the induction of PDX1 was further enhanced 60-fold over RAalone (see FIG. 37A). This very robust PDX1 induction was greater than1400-fold higher than with no activin or RA/FGF treatment.Interestingly, addition of FGF-4 and FGF-10 simultaneously abolished thebeneficial effect of the FGF-10, producing only the modest PDX1 increaseattributed to FGF-4 addition.

Addition of RA/FGF-4 or RA/FGF-10 combinations did not increase theexpression of marker genes not associated with foregut endoderm whencompared to cells not exposed to RA/FGF combinations (see FIG. 37B-C).

Example 14 Retinoic Acid Dose Affects Anterior-Posterior (A-P) PositionIn Vitro

To determine whether the dose of RA affects A-P position in in vitrocell cultures, the following experiment was performed.

Human embryonic stem cells were cultured with or without activins forfour days. On day four, FGF-10 at 50 ng/ml was added to the culture incombination with RA at 0.04.4 μM, 0.2 μM or 1.0 μM. The expression ofthe PDX1 marker gene as well as other markers not specific for foregutendoderm were quantitated by Q-PCR.

The addition of retinoic acid at various doses, in combination withFGF-10 at 50 ng/ml, induced differential gene expression patterns thatcorrelate with specific anterior-posterior positional patterns. Thehighest dose of RA (1 μM) preferentially induced expression of anteriorendoderm marker (HOXA3) and also produced the most robust increase inPDX1 (FIG. 38A-B). The middle dose of RA (0.2 μM) induced midgutendoderm markers (CDX1, HOXC6) (see FIG. 38C and 41E), while the lowestdose of RA (0.04 μM) preferentially induced a marker of hindgut endoderm(HOXA13) (see FIG. 38D). The RA dose had essentially no effect on therelative expression of either neural (SOX1) or neuronal (NFM) markers(see FIG. 38F-G). This example highlights the use of RA as a morphogenin vitro and in particular as a morphogen of endoderm derivatives ofdifferentiating hESCs.

Example 15 Use of B27 Supplement Enhances Expression of PDX1

PDX1 expression in definitive endoderm can be influenced by the use of anumber of factors and cell growth/differentiation conditions. In thefollowing experiment, we show that the use of B27 supplement enhancesthe expression of PDX1 in definitive endoderm cells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hES cells grown on mouseembryonic fibroblast feeders with high dose activin A (100-200 ng/ml in0.5-2% FBS/DMEM/F12) for 4 days. The no activin A control received0.5-2% FBS/DMEM/F12 with no added activin A. At four days, culturesreceived either no activin A in 2% FBS (none), and in 2% serumreplacement (SR), or 50 ng/ml activin A together with 2 μM RA and 50ng/ml FGF-10 in 2% FBS/DMEM/F12 (none, +FBS, +B27) and similarly in 2%Serum replacement (SR). B27 supplement, (Gibco/BRL), was added as a 1/50dilution directly into 2% FBS/DMEM/F12 (+B27). Duplicate cell sampleswhere taken for each point, and total RNA was isolated and subjected toQ-PCR as previously described.

FIG. 39A-E shows that serum-free supplement B27 provided an additionalbenefit for induction of PDX1 gene expression without inducing anincrease in the expression of markers genes not specific for foregutendoderm as compared to such marker gene expression in cells grownwithout serum.

Example 16 Use of Activin B to Enhance Induction of PDX1

This Example shows that the use of activin B enhances thedifferentiation of PDX1-negative cells to PDX1 -positive cells in invitro cell culture.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (50 ng/ml) in low serum/RPMIfor 6 days. The FBS dose was 0% on day one, 0.2% on day two and 2% ondays 3-6. The negative control for definitive endoderm production (NF)received 2% FBS/RPMI with no added activin A. In order to induce PDX1expression, each of the cultures received retinoic acid at 2 μM in 2%FBS/RPMI on day 6. The cultures treated with activin A on days onethrough five were provided with different dosing combinations of activinA and activin B or remained in activin A alone at 50 ng/ml. The noactivin A control culture (NF) was provided neither activin A noractivin B. This RA/activin treatment was carried out for 3 days at whichtime PDX1 gene expression was measured by Q-PCR from duplicate cellsamples.

FIG. 40A shows that the addition of activin B at doses ranging from10-50 ng/ml (a10, a25 and a5O) in the presence of 25 ng/ml (A25) or 50ng/ml (A50) of activin A increased the PDX1 expression at least 2-foldover the culture that received only activin A at 50 ng/ml. The increasein PDX1 as a result of activin B addition was without increase in HNF6expression (see FIG. 40B), which is a marker for liver as well aspancreas at this time in development. This result suggests that theproportion of cells differentiating to pancreas had been increasedrelative to liver.

Example 17 Use of Serum Dose to Enhance Induction of PDX1

The expression of PDX1 in definitive endoderm cells is influenced by theamount of serum present in the cell culture throughout thedifferentiation process. The following experiment shows that the levelof serum in a culture during the differentiation of hESCs toPDX1-negative definitive endoderm has an effect on the expression ofPDX1 during further differentiation of these cells to PDX1-positiveendoderm.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.1% on day one, 0.5% on day twoand either 0.5%, 2% or 10% on days 3-5. The no activin A control (NF)received the same daily FBS/RPMI dosing, but with no added activin A.PDX1 expression was induced beginning at day 6 by the addition of RA.During days 6-7, cultures received retinoic acid at 2 μM in 0.5%FBS/RPMI, 1 μM on day 8 and 0.2 μM on day 9-11. The activin A waslowered to 50 ng/ml during retinoic acid treatment and was left absentfrom the no activin A control (NF).

FIG. 41A shows that the FBS dosing during the 3 day period of definitiveendoderm induction (days 3, 4 and 5) had a lasting ability to change theinduction of PDX1 gene expression during the retinoic acid treatment.This was without significant alteration in the expression pattern ofZIC1 (FIG. 41B) or SOX7 (FIG. 41C) gene expression.

Example 18 Use of Conditioned Medium to Enhance Induction of PDX1

Other factors and growth conditions which influence the expression ofPDX1 in definitive endoderm cells were also studied. The followingexperiment shows the effect of conditioned media on the differentiationof PDX1-negative definitive endoderm cells to PDX1 -positive endodermcells.

Human embryonic stem cells were induced to differentiate to definitiveendoderm by treatment of undifferentiated hESCs grown on mouse embryonicfibroblast feeders with high dose activin A (100 ng/ml) in lowserum/RPMI for 5 days. The FBS dose was 0.2% on day one, 0.5% on day twoand 2% on days 3-5.

The definitive endoderm cultures generated by 5 days of activin Atreatment were then induced to differentiate to PDX1 expressing endodermby the addition of RA in 2% FBS/RPMI containing activin A at 25 ng/mlfor four days. The RA was 2 μM for the first two days of addition, 1 μMon the third day and 0.5 μM on the fourth day. This base medium for PDX1induction was provided fresh (2A25R) or after conditioning for 24 hoursby one of four different cell populations. Conditioned media (CM) weregenerated from either mouse embryonic fibroblasts (MEFCM) or from hESCsthat were first differentiated for 5 days by one of three conditions; i)3% FBS/RPMI (CM2), or ii) activin A (CM3) or iii) bone morphogenicprotein 4 (BMP4) (CM4). Activin A or BMP4 factors were provided at 100ng/ml under the same FBS dosing regimen described above (0.2%, 0.5%,2%). These three different differentiation paradigms yield three verydifferent populations of human cells by which the PDX1 induction mediacan be conditioned. The 3% FBS without added growth factor (NF) yields aheterogeneous population composed in large part of extraembryonicendoderm, ectoderm and mesoderm cells. The activin A treated culture(A100) yields a large proportion of definitive endoderm and the BMP4treated culture (B100) yields primarily trophectoderm and someextraembryonic endoderm.

FIG. 42A shows that PDX1 was induced equivalently in fresh andconditioned media over the first two days of RA treatment. However, bythe third day PDX1 expression had started to decrease in fresh media andMEF conditioned media treatments. The differentiated hESCs producedconditioned media that resulted in maintenance or further increases inthe PDX1 gene expression at levels 3 to 4-fold greater than fresh media.The effect of maintaining high PDX1 expression in hESC-conditioned mediawas further amplified on day four of RA treatment achieving levels 6 to7-fold higher than in fresh media. FIG. 42B shows that the conditionedmedia treatments resulted in much lower levels of CDX1 gene expression,a gene not expressed in the region of PDX1 expressing endoderm. Thisindicates that the overall purity of PDX1-expressing endoderm was muchenhanced by treating definitive endoderm with conditioned mediagenerated from differentiated hESC cultures.

FIG. 43 shows that PDX1 gene expression exhibited a positive doseresponse to the amount of conditioned media applied to the definitiveendoderm cells. Total volume of media added to each plate was 5 ml andthe indicated volume (see FIG. 43) of conditioned media was diluted intofresh media (A25R). It is of note that just 1 ml of conditioned mediaadded into 4 ml of fresh media was still able to induce and maintainhigher PDX1 expression levels than 5 ml of fresh media alone. Thissuggests that the beneficial effect of conditioned media for inductionof PDX1 expressing endoderm is dependent on the release of somesubstance or substances from the cells into the conditioned media andthat this substance(s) dose dependently enhances production of PDX1-expressing endoderm.

Example 19 Validation of Antibodies which Bind to PDX1

Antibodies that bind to PDX1 are useful tools for monitoring theinduction of PDX1 expression in a cell population. This Example showsthat rabbit polyclonal and IgY antibodies to PDX1 can be used to detectthe presence of this protein.

In a first experiment, IgY anti-PDX1 (IgY α-PDX1) antibody binding toPDX1 in cell lysates was validated by Western blot analysis. In thisanalysis, the binding of IgY α-PDX1 antibody to 50 μg of total celllysate from MDX12 human fibroblasts or MDX12 cells transfected 24 hrspreviously with a PDX1 expression vector was compared. The cell lysatesseparated by SDS-PAGE, transferred to a membrane by electroblotting, andthen probed with the IgY α-PDX1 primary antiserum followed by alkalinephosphatase conjugated rabbit anti-IgY (Rb α-IgY) secondary antibodies.Different dilutions of primary and secondary antibodies were applied toseparate strips of the membrane in the following combinations: A (500×dilution of primary, 10,000× dilution of secondary), B (2,000×,10,000×), C (500×, 40,000×), D (2,000×, 40,000), E (8,000×, 40,000×).

Binding was detected in cells transfected with the PDX1 expressionvector (PDX1-positive) at all of the tested antibody combinations.Binding was only observed in untransfected (PDX1-negative) fibroblastswhen using the highest concentrations of both primary and secondaryantibody together (combination A). Such non-specific binding wascharacterized by the detection of an additional band at a molecularweight slightly higher than PDX1 in both the transfected anduntransfected fibroblasts.

In a second experiment, the binding of polyclonal rabbit anti-PDX1 (Rbα-PDX1) antibody to PDX1 was tested by immunocytochemistry. To produce aPDX1 expressing cell for such experiments, MS1-V cells (ATCC # CRL-2460)were transiently transfected with an expression vector of PDX1-EGFP(constructed using pEGFP-N1, Clontech). Transfected cells were thenlabeled with Rb α-PDX1 and α-EGFP antisera. Transfected cells werevisualized by both EGFP fluorescence as well as a-EGFPimmunocytochemistry through the use of a Cy5 conjugated secondaryantibody. PDX1 immunofluorescence was visualized through the use of anα-Rb Cy3-conjugated secondary antibody.

Binding of the Rb α-PDX1 and the α-EGPF antibodies co-localized with GPFexpression.

Example 20 Immunocytochemistry of Human Pancreatic Tissue

This Example shows that antibodies having specificity for PDX1 can beused to identify human PDX1-positive cells by immunocytochemistry.

In a first experiment, paraffin embedded sections of human pancreas werestained for insulin with guinea pig anti-insulin (Gp a-Ins) primaryantibody at a 1/200 dilution followed by dog anti-guinea pig (D a-Gp)secondary antibody conjugated to Cy2 at a 1/100 dilution. In a secondexperiment, the same paraffin embedded sections of human pancreas werestained for PDX1 with IgY a-PDX1 primary antibody at a 1/4000 dilutionfollowed Rb a-IgY secondary antibody conjugated to AF555 at a 1/300dilution. The images collected from the first and second experimentswhere then merged. In a third experiment, cells that were stained withIgY a-PDX1 antibodies were also stained with DAPI.

Analysis of the human pancreatic sections revealed the presence ofstrong staining of islets of Langerhans. Although the strongest PDX1signal appeared in islets (insulin-positive), weak staining was alsoseen in acinar tissue (insulin-negative). DAPI and PDX1 co-stainingshows that PDX1 was mostly but not exclusively localized to the nucleus.

Example 21 Immunoprecipitation of PDX1 from Retinoic Acid Treated Cells

To further confirm PDX1 expression in definitive endoderm cells thathave been differentiated in the presence of RA and the lack of PDX1 indefinitive endoderm cells that have not been differentiated with RA, arabbit anti-PDX1 (Rb a-PDX1) antibody was used to immunoprecipitate PDX1from both RA differentiated and undifferentiated definitive endodermcells. Immunoprecipitated RA was detected by Western blot analysis usingIgY a-PDX1 antibody.

To obtain undifferentiated and differentiated definitive endoderm celllysates for immunoprecipitation, hESCs were treated for 5 days withactivin A at 100 ng/ml in low serum (definitive endoderm) followed bytreatment with activin A at 50 ng/ml and 2 μM all-trans RA for two days,1 μM for one day and 0.2 μM for one day (PDX1-positive foregutendoderm). As a positive control cell lysates were also prepared fromMS1-V cells (ATCC # CRL-2460) transfected with a PDX1 expression vector.PDX1 was immunoprecipitated by adding Rb a-PDX1 and rabbit-specificsecondary antibodies to each lysate. The precipitate was harvested bycentrifugation. Immunoprecipitates were dissolved in SDS-containingbuffer then loaded onto a polyacrylamide gel. After separation, theproteins were transferred to a membrane by electroblotting, and thenprobed with the IgY α-PDX1 primary antibody followed by labeled Rb α-IgYsecondary antibodies.

Immunoprecipitates collected from the MS1-V positive control cells aswell as those from day 8 (lane d8, three days after the start of RAtreatment) and day 9 (lane d9, four days after the start of RA) cellswere positive for PDX1 protein (FIG. 44). Precipitates obtained fromundifferentiated definitive endoderm cells (that is, day 5 cells treatedwith activin A—designated (A) in FIG. 44) and undifferentiated hESCs(that is, untreated day 5 cells—designated as (NF) in FIG. 44) werenegative for PDX1.

Example 22 Generation of PDX1 Promoter-EGFP Transgenic hESC Lines

In order to use the PDX1 marker for cell isolation, we geneticallytagged PDX1-positive foregut endoderm cells with an expressible reportergene. This Example describes the construction of a vector comprising areporter cassette which comprises a reporter gene under the control ofthe PDX1 regulatory region. This Example also describes the preparationof a cell, such as a human embryonic stem cell, transfected with thisvector as well as a cell having this reporter cassette integrated intoits genome.

PDX1-expressing definitive endoderm cell lines genetically tagged with areporter gene were constructed by placing a GFP reporter gene under thecontrol of the regulatory region (promoter) of the PDX1 gene. First, aplasmid construct in which EGFP expression is driven by the human PDX1gene promoter was generated by replacing the CMV promoter of vectorpEGFP-N1 (Clontech) with the human PDX1 control region (GenbankAccession No. AF192496, the disclosure of which is incorporated hereinby reference in its entirety), which comprises a nucleotide sequenceranging from about 4.4 kilobase pairs (kb) upstream to about 85 basepairs (bp) downstream of the PDX1 transcription start site. This regioncontains the characterized regulatory elements of the PDX1 gene, and itis sufficient to confer the normal PDX1 expression pattern in transgenicmice. In the resulting vector, expression of EFGP is driven by the PDX1promoter. In some experiments, this vector can be transfected intohESCs.

The PDX1 promoter/EGFP cassette was excised from the above vector, andthen subcloned into a selection vector containing the neomycinphosphotransferase gene under control of the phosphoglycerate kinase-1promoter. The selection cassette was flanked by flp recombinaserecognition sites to allow removal of the cassette. This selectionvector was linearized, and then introduced into hESCs using standardlipofection methods. Following 10-14 days of selection in G418,undifferentiated transgenic hESC clones were isolated and expanded.

Example 23 Isolation of PDX1-Positive Foregut Endoderm

The following Example demonstrates that hESCs comprising the PDX1promoter/EGFP cassette can be differentiated into PDX1-positive endodermcells and then subsequently isolated by fluorescence-activated cellsorting (FACS).

PDX1 promoter/EGFP transgenic hESCs were differentiated for 5 days inactivin A-containing media followed by two days in media comprisingactivin A and RA. The differentiated cells were then harvested bytrypsin digestion and sorted on a Becton Dickinson FACS Diva directlyinto RNA lysis buffer or PBS. A sample of single live cells was takenwithout gating for EGFP (Live) and single live cells were gated intoEGFP positive (GFP) and GFP negative (Neg) populations. In oneexperiment, the EGFP positive fraction was separated into two equallysized populations according to fluorescence intensity (Hi and Lo).

Following sorting, cell populations were analyzed by both Q-PCR andimmunocytochemistry. For Q-PCR analysis, RNA was prepared using QiagenRNeasy columns and then converted to cDNA. Q-PCR was conducted asdescribed previously. For immunocytochemistry analysis, cells weresorted into PBS, fixed for 10 minutes in 4% paraformaldehyde, andadhered to glass slides using a Cytospin centrifuge. Primary antibodiesto Cytokeratin19 (KRT19) were from Chemicon; to Hepatocyte nuclearfactor 3 beta (HNF3β) from Santa Cruz; to Glucose Transporter 2 (GLUT2)from R&D systems. Appropriate secondary antibodies conjugated to FITC(green) or Rhodamine (Red) were used to detect binding of the primaryantibodies.

A typical FACS sort of differentiated cells is shown in FIG. 45. Thepercent isolated PDX1-positive cells in this example was approximately7%, which varied depending on the differentiation efficiency from about1% to about 20%.

Sorted cells were further subjected to Q-PCR analysis. Differentiatedcells showed a correlation of EGFP fluorescence with endogenous PDX1gene expression. Compared to non-fluorescing cells, the EGFP positivecells showed a greater than 20-fold increase in PDX1 expression levels(FIG. 46). The separation of high and low EGFP intensity cells indicatedthat EGFP expression level correlated with PDX1 expression level (FIG.47). In addition to PDX1 marker analysis, sorted cells were subjected toQ-PCR analysis of several genes that are expressed in pancreaticendoderm. Products of each of these marker genes (NKX2.2, GLUT2, KRT19,HNF4α and HNF3β) were all enriched in the EGFP positive fraction (FIGS.48A-E). In contrast, the neural markers ZIC1 and GFAP were not enrichedin sorted EGFP expressing cells (FIGS. 49A and B).

By immunocytochemistry, virtually all the isolated PDX1-positive cellswere seen to express KRT19 and GLUT2. This result is expected for cellsof the pancreatic endoderm lineage. Many of these cells were also HNF3βpositive by antibody staining.

Example 24 Transplantation of Human Definitive Endoderm Cells UnderMouse Kidney Capsule

To demonstrate that the human definitive endoderm cells produced usingthe methods described herein are capable of responding todifferentiation factors so as to produce cells that are derived from thegut tube, such human definitive endoderm cells were subjected to an invivo differentiation protocol.

Human definitive endoderm cells were produced as described in theforegoing Examples. Such cells were harvested and transplanted under thekidney capsule of immunocompromised mice using standard procedures.After three weeks, the mice were sacrificed and the transplanted tissuewas removed, sectioned and subjected to histological andimmunocytochemical analysis.

FIGS. 50A-D show that after three weeks post-transplantation, the humandefinitive endoderm cells differentiated into cells and cellularstructures derived from the gut tube. In particular, FIG. 50A showshematoxylin and eosin stained sections of transplanted human definitiveendoderm tissue that has differentiated into gut-tube-like structures.FIG. 50B shows a transplanted human definitive endoderm sectionimmunostained with antibody to hepatocyte specific antigen (HSA). Thisresult indicates that the human definitive endoderm cells are capable ofdifferentiating into liver or liver precursor cells. FIGS. 50C and 50Dshow a transplanted human definitive endoderm section immunostained withantibody to villin and antibody to caudal type homeobox transcriptionfactor 2 (CDX2), respectively. These results indicate that the humandefinitive endoderm cells are capable of differentiating into intestinalcells or intestinal cell precursors.

Example 25 Identification of Differentiation Factors Capable ofPromoting the Differentiation of Human Definitive Endoderm Cells InVitro

To exemplify the differentiation factor screening methods describedherein, populations of human definitive endoderm cells produced usingthe methods described herein were separately provided with severalcandidate differentiation factors while determining the normalizedexpression levels of certain marker gene products at various timepoints.

Human definitive endoderm cells were produced as described in theforegoing Examples. In brief, hESCs cells were grown in the presence of100 ng/ml activin A in low serum RPMI medium for four days, wherein thefetal bovine serum (FBS) concentration on day 1 was 0%, on day 2 was0.2% and on days 3-4 was 2%. After formation of definitive endoderm,beginning on day 5 and ending on day 10, cell populations maintained inindividual plates in RPMI containing 0.2% FBS were treated with one of:Wnt3B at 20 ng/ml, FGF2 at 5 ng/ml or FGF2 at 100 ng/ml. The expressionof marker gene products for albumin, PROX1 and TITF1 were quantitatedusing Q-PCR.

FIG. 51A shows that expression of the albumin gene product (a marker forliver precursors and liver cells) substantially increased on days 9 and10 in response to FGF2 at 5 ng/ml as compared to expression indefinitive endoderm cells on day 4 prior to treatment with thisdifferentiation factor. Expression of the albumin gene product was alsoincreased in response to 20 ng/ml Wnt3B on days 9 and 10 as compared toexpression in untreated definitive endoderm cells, however, the increasewas not as large as that observed for the 5 ng/ml FGF2 treatment. Ofparticular significance is the observation that the expression of thealbumin gene product was not increased on days 9 and 10 in response toFGF2 at 100 ng/ml as compared to expression in definitive endoderm cellson day 4. Similar results were seen with the PROX1 marker (a secondmarker for liver precursors and liver cells) as shown in FIG. 51B. FIG.51C shows that in cell populations provided with 100 ng/ml FGF2,expression of the TITF1 marker gene substantially increased on days 7, 9and 10 as compared to expression in definitive endoderm cells on day 4prior to treatment with this differentiation factor, but FGF2 at 5 ng/mlhad very little effect on expression of this gene product as compared tountreated definitive endoderm. Taken together, the results shown inFIGS. 51A-C indicate that the concentration at which the candidatedifferentiation factor is provided to the cell population can affect thedifferentiation fate of definitive endoderm cells in vitro.

Example 26 Marker Upregulation and Downregulation in Response toCandidate Differentiation Factors

To further exemplify the differentiation factor screening methodsdescribed herein, populations of human definitive endoderm cells werescreened with candidate differentiation factors using procedures similarto those described in Example 25.

Human definitive endoderm cells were produced as described in theforegoing Examples. In brief, hESCs cells were grown in the presence of100 ng/ml activin A in low serum RPMI medium for four days, wherein thefetal bovine serum (FBS) concentration on day 1 was 0%, on day 2 was0.2% and on days 3-4 was 2%. After formation of definitive endoderm,beginning on day 5 and ending on day 10, cell populations maintained inindividual plates in RPMI containing 0.2% FBS were treated with one of:Wnt3A at 20-50 ng/ml, FGF2 at 5 ng/ml or FGF2 at 100 ng/ml. On day 5post definitive endoderm formation (day 9 after the start of thedifferentiation from hESCs), BMP4 was added to all the cultures at aconcentration of 50 ng/ml. The expression of marker gene products(mRNAs) for alpha fetoprotein (AFP), cytochrome P450 7A (CYP7A),tyrosine aminotransferase (TAT), hepatocyte nuclear factor 4a (HNF4a),CXC-type chemokine receptor 4 (CXCR4), von Willebrand factor (VWF),vascular cell adhesion molecule-1 (VACM1), apolipoprotein Al (APOA1),glucose transporter-2 (GLUT2), alpha-1-antitrypsin (AAT), glukokinase(GLUKO), and human hematopoietically expressed homeobox (hHEX) werequantitated using Q-PCR.

FIGS. 52A-B show that expression of the AFP gene product (a marker forliver precursors and liver cells) and AAT substantially increased ondays 9 and 10 in response to FGF2 at 5 ng/ml and BMP4 at 50 ng/ml ascompared to expression in definitive endoderm cells on day 4. Expressionof AFP and AAT mRNAs was not substantially increased by higherconcentration of FGF2 (100 ng/ml) even in the presence of BMP4 (FIGS.51A-B days 9 and 10). In contrast to the above results, the expressionof GLUKO, hHEX and TAT mRNAs was substantially upregulated in thepresence of FGF2 at 100 ng/ml and BMP4 at 50 ng/ml on days 9 and 10 ascompared to expression in definitive endoderm cells on day 4. In thecase of GLUKO, neither Wnt3A nor FGF2 at 5 ng/ml with or without BMP4caused an increase in the expression of this marker (FIG. 52C). FGF2 at5 ng/ml did, however, cause an increase in expression of hHEX in thepresence of BMP to an extent greater than or equal to the increasecaused by FGF2 at 100 ng/ml in the presence of BMP (FIG. 52D).Expression of TAT on days 9 and 10 as compared to expression indefinitive endoderm cells was increased by each of the factors tested(FIG. 52E). Additionally, certain cell markers were expressed at anincreased level as compared to definitive endoderm cells in the presenceof Wnt3A, but not in response to FGF/BMP combinations. In particular,the expression of hNF4a mRNA significantly increased on days 9 and 10 inresponse to the combination of Wnt3A and BMP4 (FIG. 52F). Furthermore,CYP7A showed a marginal increase on response to Wnt3A/BMP4 on day 10(FIG. 52G).

Several markers that are known to be expressed in a number of differentcells types were also observed. Specifically the markers APOA1, GLUT2,VCAM1, VWF and CXCR4 were examined. Previously the expression of each ofthese markers has been correlated with specific cell types as follows:The markers APOA1 and GLUT2 are highly expressed in the liver andmoderately expressed in the duodenum and small intestine. The markerVCAM1 is expressed at a high level in the liver, expressed at a moderatelevel in the stomach, duodenum, and small intestine, and expressed atlower but significant levels in the lung and pancreas. In contrast, themarkers VWF and CXCR4 are expressed at high levels in the lung but onlyat low levels in liver. Both VWF and CXCR4 are also expressed atmoderate to high levels in the stomach, pancreas, duodenum, and smallintestine.

Expression of each of the above-described markers was monitored indefinitive endoderm cell cultures contacted with combinations of Wnt3A,FGF2 and BMP4. Consistent with the above results, FIGS. 52H-J show thatGLUT2, APOA1 and VCAM1 mRNA expression was increased in response to thecombination of FGF2 at 5 ng/ml and BMP4 on days 9 and 10 as compared tothe expression in definitive endoderm. The mRNA expression for thesemarkers was not substantially increased in response to the combinationof FGF2 at 100 ng/ml an BMP4. In the case of the APOA1 and VCAM1 markermRNAs, the largest increase in expression on days 9 and 10 was mediatedby the combination of Wnt3A and BMP4 (FIGS. 52I-J).

In addition to the foregoing, the expression of certain mRNAs wasdecreased as compared to the expression in definitive endoderm. Forexample, as compared to the expression in definitive endoderm, both VWFand CXCR4 mRNA expression was decreased after contact with Wnt3A in thepresence and in the absence of BMP4 as well as after contact with FGF2at 5 ng/ml in the presence and in the absence of BMP4 (FIGS. 52K-L).Contact with FGF2 at 100 ng/ml, both in the absence and and in thepresence of BMP4, greatly slowed the rate of decrease of these twomarkers (FIGS. 52K-L). In fact, expression of CXCR4 was substantiallymaintained even on day 10 (FIG. 52L).

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, by thephrase “consisting essentially of” is meant including any elementslisted after the phrase, and limited to other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

REFERENCES

Numerous literature and patent references have been cited in the presentpatent application. Each and every reference that is cited in thispatent application is incorporated by reference herein in its entirety.

For some references, the complete citation is in the body of the text.For other references the citation in the body of the text is by authorand year, the complete citation being as follows:

Alexander, J., Rothenberg, M., Henry, G. L., and Stainier, D. Y. (1999).Casanova plays an early and essential role in endoderm formation inzebrafish. Dev Biol 215, 343-357.

Alexander, J., and Stainier, D. Y. (1999). A molecular pathway leadingto endoderm formation in zebrafish. Curr Biol 9,1147-1157.

Aoki, T. O., Mathieu, J., Saint-Etienne, L., Rebagliati, M. R.,Peyrieras, N., and Rosa, F. M. (2002). Regulation of nodal signallingand mesendoderm formation by TARAM-A, a TGFbeta-related type I receptor.Dev Biol 241, 273-288.

Beck, S., Le Good, J. A., Guzman, M., Ben Haim, N., Roy, K., Beermann,F., and Constam, D. B. (2002). Extra-embryonic proteases regulate Nodalsignalling during gastrulation. Nat Cell Biol 4, 981-985.

Beddington, R. S., Rashbass, P., and Wilson, V. (1992). Brachyury—a geneaffecting mouse gastrulation and early organogenesis. Dev Suppl,157-165.

Bongso, A., Fong, C. Y., Ng, S. C., and Ratnam, S. (1994). Isolation andculture of inner cell mass cells from human blastocysts. Hum Reprod 9,2110-2117.

Chang, H., Brown, C. W., and Matzuk, M. M. (2002). Genetic analysis ofthe mammalian transforming growth factor-beta superfamily. Endocr Rev23, 787-823.

Conlon, F. L., Lyons, K. M., Takaesu, N., Barth, K. S., Kispert, A.,Herrmann, B., and Robertson, E. J. (1994). A primary requirement fornodal in the formation and maintenance of the primitive streak in themouse. Development 120, 1919-1928.

Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F., and Talbot, W.S. (2003). The role of the zebrafish nodal-related genes squint andcyclops in patterning of mesendoderm. Development 130, 1837-1851.

Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G.,Sirotkin, H. I., Schier, A. F., and Talbot, W. S. (1998). Zebrafishorganizer development and germ-layer formation require nodal-relatedsignals. Nature 395, 181-185.

Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996). HIV-1entry cofactor: functional cDNA cloning of a seven-transmembrane, Gprotein-coupled receptor. Science 272, 872-877.

Futaki, S., Hayashi, Y., Yamashita, M., Yagi, K., Bono, H., Hayashizaki,Y., Okazaki, Y., and Sekiguchi, K. (2003). Molecular basis ofconstitutive production of basement membrane components: Gene expressionprofiles of engelbreth-holm-swarm tumor and F9 embryonal carcinomacells. J Biol Chem.

Grapin-Botton, A., and Melton, D. A. (2000). Endoderm development: frompatterning to organogenesis. Trends Genet 16, 124-130.

Harris, T. M., and Childs, G. (2002). Global gene expression patternsduring differentiation of F9 embryonal carcinoma cells into parietalendoderm. Funct Integr Genomics 2, 105-119.

Hogan, B. L. (1996). Bone morphogenetic proteins in development. CurrOpin Genet Dev 6, 432-438.

Hogan, B. L. (1997). Pluripotent embryonic cells and methods of makingsame (U.S.A., Vanderbilt University).

Howe, C. C., Overton, G. C., Sawicki, J., Solter, D., Stein, P., andStrickland, S. (1988). Expression of SPARC/osteonectin transcript inmurine embryos and gonads. Differentiation 37, 20-25.

Hudson, C., Clements, D., Friday, R. V., Stott, D., and Woodland, H. R.(1997). Xsox17alpha and -beta mediate endoderm formation in Xenopus.Cell 91, 397-405.

Imada, M., Imada, S., Iwasaki, H., Kume, A., Yamaguchi, H., and Moore,E. E. (1987). Fetomodulin: marker surface protein of fetal developmentwhich is modulatable by cyclic AMP. Dev Biol 122, 483-491.

Kanai-Azuma, M., Kanai, Y., Gad, J. M., Tajima, Y., Taya, C., Kurohmaru,M., Sanai, Y., Yonekawa, H., Yazaki, K., Tam, P. P., and Hayashi, Y.(2002). Depletion of definitive gut endoderm in Sox17-null mutant mice.Development 129, 2367-2379.

Katoh, M. (2002). Expression of human SOX7 in normal tissues and tumors.Int J Mol Med 9,363-368.

Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron, S., Yelon,D., Thisse, B., and Stainier, D. Y. (2001). casanova encodes a novelSox-related protein necessary and sufficient for early endodermformation in zebrafish. Genes Dev 15, 1493-1505.

Kim, C. H., and Broxmeyer, H. E. (1999). Chemokines: signal lamps fortrafficking of T and B cells for development and effector function. JLeukoc Biol 65, 6-15.

Kimelman, D., and Griffin, K. J. (2000). Vertebrate mesendoderminduction and patterning. Curr Opin Genet Dev 10, 350-356.

Kubo A, Shinozaki K, Shannon J M, Kouskoff V, Kennedy M, Woo S, FehlingH J, Keller G. (2004) Development of definitive endoderm from embryonicstem cells in culture. Development. 131,1651-62.

Kumar, A., Novoselov, V., Celeste, A. J., Wolfman, N. M., ten Dijke, P.,and Kuehn, M. R. (2001). Nodal signaling uses activin and transforminggrowth factor-beta receptor-regulated Smads. J Biol Chem 276, 656-661.

Labosky, P. A., Barlow, D. P., and Hogan, B. L. (1994a). Embryonic germcell lines and their derivation from mouse primordial germ cells. CibaFound Symp 182, 157-168; discussion 168-178.

Labosky, P. A., Barlow, D. P., and Hogan, B. L. (1994b). Mouse embryonicgerm (EG) cell lines: transmission through the germline and differencesin the methylation imprint of insulin-like growth factor 2 receptor(Igf2r) gene compared with embryonic stem (ES) cell lines. Development120, 3197-3204.

Lickert, H., Kutsch, S., Kanzler, B., Tamai, Y., Taketo, M. M., andKemler, R. (2002). Formation of multiple hearts in mice followingdeletion of beta-catenin in the embryonic endoderm. Dev Cell 3, 171-181.

Lu, C. C., Brennan, J., and Robertson, E. J. (2001). From fertilizationto gastrulation: axis formation in the mouse embryo. Curr Opin Genet Dev11, 384-392.

Ma, Q., Jones, D., and Springer, T. A. (1999). The chemokine receptorCXCR4 is required for the retention of B lineage and granulocyticprecursors within the bone marrow microenvironment. Immunity 10,463-471.

McGrath K E, Koniski A D, Maltby K M, McGann J K, Palis J. (1999)Embryonic expression and function of the chemokine SDF-1 and itsreceptor, CXCR4. Dev Biol. 213, 442-56.

Miyazono, K., Kusanagi, K., and Inoue, H. (2001). Divergence andconvergence of TGF-beta/BMP signaling. J Cell Physiol 187, 265-276.

Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S.,Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996).Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in micelacking the CXC chemokine PBSF/SDF-1. Nature 382, 635-638.

Niwa, H. (2001). Molecular mechanism to maintain stem cell renewal of EScells. Cell Struct Funct 26, 137-148.

Ogura, H., Aruga, J., and Mikoshiba, K. (2001). Behavioral abnormalitiesof Zic1 and Zic2 mutant mice: implications as models for humanneurological disorders. Behav Genet 31, 317-324.

Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., and Bongso, A.(2000). Embryonic stem cell lines from human blastocysts: somaticdifferentiation in vitro. Nat Biotechnol 18, 399-404.

Rodaway, A., and Patient, R. (2001). Mesendoderm. an ancient germ layer?Cell 105, 169-172.

Rodaway, A., Takeda, H., Koshida, S., Broadbent, J., Price, B., Smith,J. C., Patient, R., and Holder, N. (1999). Induction of the mesendodermin the zebrafish germ ring by yolk cell-derived TGF-beta family signalsand discrimination of mesoderm and endoderm by FGF. Development 126,3067-3078.

Rohr, K. B., Schulte-Merker, S., and Tautz, D. (1999). Zebrafish zic1expression in brain and somites is affected by BMP and hedgehogsignalling. Mech Dev 85, 147-159.

Schier, A. F. (2003). Nodal signaling in vertebrate development. AnnuRev Cell Dev Biol 19, 589-621.

Schoenwolf, G. C., and Smith, J. L. (2000). Gastrulation and earlymesodermal patterning in vertebrates. Methods Mol Biol 135, 113-125.

Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M., Littlefield, J.W., Donovan, P. J., Blumenthal, P. D., Huggins, G. R., and Gearhart, J.D. (1998). Derivation of pluripotent stem cells from cultured humanprimordial germ cells. Proc Natl Acad Sci U S A 95, 13726-13731.

Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E.,Warnock, G. L., Kneteman, N. M., and Rajotte, R. V. (2000). Islettransplantation in seven patients with type 1 diabetes mellitus using aglucocorticoid-free immunosuppressive regimen. N Engl J Med 343,230-238.

Shapiro, A. M., Ryan, E. A., and Lakey, J. R. (2001a). Pancreatic islettransplantation in the treatment of diabetes mellitus. Best Pract ResClin Endocrinol Metab 15, 241-264.

Shapiro, J., Ryan, E., Warnock, G. L., Kneteman, N. M., Lakey, J.,Korbutt, G. S., and Rajotte, R. V. (2001b). Could fewer islet cells betransplanted in type 1 diabetes? Insulin independence should be dominantforce in islet transplantation. Bmj 322, 861.

Shiozawa, M., Hiraoka, Y., Komatsu, N., Ogawa, M., Sakai, Y., and Aiso,S. (1996). Cloning and characterization of Xenopus laevis xSox7 cDNA.Biochim Biophys Acta 1309, 73-76.

Smith, J. (1997). Brachyury and the T-box genes. Curr Opin Genet Dev 7,474-480.

Smith, J. C., Armes, N. A., Conlon, F. L., Tada, M., Umbhauer, M., andWeston, K. M. (1997). Upstream and downstream from Brachyury, a generequired for vertebrate mesoderm formation. Cold Spring Harb Symp QuantBiol 62, 337-346.

Takash, W., Canizares, J., Bonneaud, N., Poulat, F., Mattei, M. G., Jay,P., and Berta, P. (2001). SOX7 transcription factor: sequence,chromosomal localisation, expression, transactivation and interferencewith Wnt signalling. Nucleic Acids Res 29, 4274-4283.

Taniguchi, K., Hiraoka, Y., Ogawa, M., Sakai, Y., Kido, S., and Aiso, S.(1999). Isolation and characterization of a mouse SRY-related cDNA,mSox7. Biochim Biophys Acta 1445, 225-231.

Technau, U. (2001). Brachyury, the blastopore and the evolution of themesoderm. Bioessays 23, 788-794.

Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A.,Swiergiel, J. J., Marshall, V. S., and Jones, J. M. (1998). Embryonicstem cell lines derived from human blastocysts. Science 282, 1145-1147.

Tremblay, K. D., Hoodless, P. A., Bikoff, E. K., and Robertson, E. J.(2000). Formation of the definitive endoderm in mouse is aSmad2-dependent process. Development 127, 3079-3090.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., DePaepe, A., and Speleman, F. (2002). Accurate normalization of real-timequantitative RT-PCR data by geometric averaging of multiple internalcontrol genes. Genome Biol 3, RESEARCH0034.

Varlet, I., Collignon, J., and Robertson, E. J. (1997). nodal expressionin the primitive endoderm is required for specification of the anterioraxis during mouse gastrulation. Development 124, 1033-1044.

Vincent, S. D., Dunn, N. R., Hayashi, S., Norris, D. P., and Robertson,E. J. (2003). Cell fate decisions within the mouse organizer aregoverned by graded Nodal signals. Genes Dev 17, 1646-1662.

Weiler-Guettler, H., Aird, W. C., Rayburn, H., Husain, M., andRosenberg, R. D. (1996). Developmentally regulated gene expression ofthrombomodulin in postimplantation mouse embryos. Development 122,2271-2281.

Weiler-Guettler, H., Yu, K., Soff, G., Gudas, L. J., and Rosenberg, R.D. (1992). Thrombomodulin gene regulation by cAMP and retinoic acid inF9 embryonal carcinoma cells. Proceedings Of The National Academy OfSciences Of The United States Of America 89, 2155-2159.

Wells, J. M., and Melton, D. A. (1999). Vertebrate endoderm development.Annu Rev Cell Dev Biol 15, 393-410.

Wells, J. M., and Melton, D. A. (2000). Early mouse endoderm ispatterned by soluble factors from adjacent germ layers. Development 127,1563-1572.

Willison, K. (1990). The mouse Brachyury gene and mesoderm formation.Trends Genet 6, 104-105.

Zhao, G. Q. (2003). Consequences of knocking out BMP signaling in themouse. Gensis 35, 43-56.

Zhou, X., Sasaki, H., Lowe, L., Hogan, B. L., and Kuehn, M. R. (1992).Nodal is a novel TGF-beta-like gene expressed in the mouse node duringgastrulation. Nature 361, 543-547.

1. A method of identifying a factor that promotes the differentiation ofhuman definitive endoderm cells, said method comprising the steps of:obtaining a human cell population comprising human definitive endodermcells, wherein said human definitive endoderm cells comprise at least10% of the human cells in said cell population; providing a candidatedifferentiation factor to said cell population; determining expressionof a marker of a cell differentiated from definitive endoderm in saidcell population at a first time point; determining expression of thesame marker in said cell population at a second time point, wherein saidsecond time point is subsequent to said first time point and whereinsaid second time point is subsequent to providing said cell populationwith said candidate differentiation factor; and determining ifexpression of the marker in said cell population at said second timepoint is increased as compared to the expression of the marker in saiddefinitive endoderm cell population at said first time point, wherein anincrease in expression of said marker in said cell population indicatesthat said candidate differentiation factor promotes the differentiationof said human definitive endoderm cells.
 2. The method of claim 1,wherein human feeder cells are present in said cell population andwherein at least 10% of the human cells other than said feeder cells aredefinitive endoderm cells.
 3. The method of claim 1, wherein said humandefinitive endoderm cells comprise at least 90% of the human cells insaid cell population.
 4. The method of claim 1, wherein said humanfeeder cells are present in said cell population and wherein at least90% of the human cells other than said feeder cells are definitiveendoderm cells.
 5. The method of claim 1, wherein said marker isselected from the group consisting of pancreatic-duodenal homeoboxfactor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6) inresponse to said candidate differentiation factor.
 6. The method ofclaim 1, wherein said marker is selected from the group consisting ofalbumin, prospero-related homeobox 1 (PROX1) and hepatocyte specificantigen (HSA) in response to said candidate differentiation factor. 7.The method of claim 1, wherein said marker is thyroid transcriptionfactor 1 (TITF1) in response to said candidate differentiation factor.8. The method of claim 1, wherein said marker is selected from the groupconsisting of villin and caudal type homeobox transcription factor 2(CDX2)) in response to said candidate differentiation factor.
 9. Themethod of claim 1, wherein said first time point is prior to providingsaid candidate differentiation factor to said cell population.
 10. Themethod of claim 1, wherein said first time point is at approximately thesame time as providing said candidate differentiation factor to saidcell population.
 11. The method of claim 1, wherein said first timepoint is subsequent to providing said candidate differentiation factorto said cell population.
 12. The method of claim 1, wherein expressionof said marker is increased.
 13. The method of claim 1, whereinexpression of said marker is determined by quantitative polymerase chainreaction (Q-PCR).
 14. The method of claim 1, wherein expression of saidmarker is determined by immunocytochemistry.
 15. The method of claim 1,wherein said candidate differentiation factor comprises a smallmolecule.
 16. The method of claim 1, wherein said candidatedifferentiation factor comprises a retinoid.
 17. The method of claim 1,wherein said candidate differentiation factor comprises retinoic acid.18. The method of claim 1, wherein said candidate differentiation factorcomprises a polypeptide.
 19. The method of claim 1, wherein saidcandidate differentiation factor comprises a growth factor.
 20. Themethod of claim 1, wherein said candidate differentiation factorcomprises FGF-10.
 21. The method of claim 1, wherein said candidatedifferentiation factor comprises FGF-2.
 22. The method of claim 1,wherein said candidate differentiation factor comprises Wnt3B.
 23. Themethod of claim 1, wherein said candidate differentiation factor is nota foregut differentiation factor.
 24. The method of claim 1, whereinsaid candidate differentiation factor is not a retinoid.
 25. The methodof claim 1, wherein said candidate differentiation factor is notretinoic acid.
 26. The method of claim 1, wherein said candidatedifferentiation factor is provided to said cell population at aconcentration of between 0.1 ng/ml to 10 mg/ml.
 27. The method of claim1, wherein said candidate differentiation factor is provided to saidcell population at a concentration of between 1 ng/ml to 1 mg/ml. 28.The method of claim 1, wherein said candidate differentiation factor isprovided to said cell population at a concentration of between 10 ng/mlto 100 μg/ml.
 29. The method of claim 1, wherein said candidatedifferentiation factor is provided to said cell population at aconcentration of between 100 ng/ml to 10 μg/ml.
 30. The method of claim1, wherein said candidate differentiation factor is provided to saidcell population at a concentration of 1 μg/ml.
 31. The method of claim1, wherein said candidate differentiation factor is provided to saidcell population at a concentration of 100 ng/ml.